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I want to label LLssLL ((2S,2'S)-N,N'-(disulfanediylbis(ethane-2,1-diyl))bis(2-((S)-2-amino-4-methylpentanamido)-4- methylpentanamide) ) with Alexa 488 NHS Ester. A280 can not be used to check the labelling effciency because there is no aromatic rings in the short peptide.
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The simplest method would be to run the peptide on HPLC before and after labeling, then measure how much of the unlabeled peptide remains by its absorbance.
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This is the protocol I use: I’m using Human Insulin and Alexa 647 NHS Ester (Succinimidyl Ester). Alexa-647 was dissolved in DMSO to a concentration of 2 mg/ml. HI was dissolved in a buffer solution at pH 8.1 to a concentration of 20 uM. 40 ul of Alexa was added to a 400 ul 20 uM HI solution. The final concentration of Alexa is about 160 uM, so the Alexa to HI ratio is about 1:8. The solution is left to react at room temperature for at least 3 hours. Afterwards I use a Sephadex G-50 or G-25 column to separate the HI-Alexa conjugated from the free dye. I collect the sample in aliquots and perform RICS to check if the conjugation has been successful, but the reaction doesn’t seem to have occurred. Do you think that the protocol is not right? Are there any mistakes in my protocol? Do you have any suggestion?
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Human insulin contains one lysine residue and 2 N-termini that can react with the dye. The pH you used is normal for this reaction. The buffer you use must not contain any amino groups, since they will react with the dye. You should use a freshly made bicarbonate buffer. The insulin must also not be in a buffer containing amino groups. If that is how it is supplied, then it should be dialyzed against bicarbonate buffer before labeling.
If your dye was not freshly dissolved just before use, it may have hydrolyzed due to water contaminating the DMSO.
The dye may have precipitated if it was added to water too quickly, before it had a chance to react. It is a good idea to add the dye a little at a time, if it has poor aqueous solubility.
The high DMSO concentration from the dye may have caused the insulin to precipitate, preventing labeling.
I don't know anything about RICS. If you want to measure the labeling stoichiometry, you should measure the dye concentration after removing the excess by using a spectrophotometer, and the insulin concentration by a protein assay (but not by UV absorbance, since the dye absorbs in the UV). You should be able to see the color of the labeled protein by eye after removing the excess dye.
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Hi all- does anyone have any research on culture in the NHS and the impact on how this impacts on any change programmes/projects?
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I think the following paper will help you out in this regard:
Change management through leadership: the mediating role of organizational culture
AA Al-Ali, SK Singh, M Al-Nahyan… - International Journal of …, 2017 - emerald.com
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I'm looking for an inexpensive source of mono-reactive Cy5 NHS ester (and/or other cyanine dyes).
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I want to conjugate a hapten via its primary amine to carboxyl groups on a carrier protein using EDC/NHS. I understand this is normally done by first activating the carboxyl-containing molecule with EDC/NHS then adding the amine-containing molecule. To avoid carrier-carrier conjugation (as it is a protein with both amine and carboxyl groups), is it possible to first activate the hapten (which has an amine but no carboxyl groups) then add it to the carrier? I seem to remember an old paper saying this is possible but cannot find a recent protocol.
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Why do you want to link your amine- containing hapten to a protein using the EDC/NHS system? What is wrong with using amine-amine cross-linking reagents? These are the usual and easy reagents for linking amine-containing haptens to proteins
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Is it possible to reverse the conjugation chemistry of EDS NHS linkage of nanoparticle surface to IgG antibody in order to reuse the particle for new conjugation of antibody? The particles i will be using have a -COOH functionalized polymer coating. Can anyone provide some publications about this? Thank you all!
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No, it's impossible with EDC/NHS chemistry. And even if you could, why would you? You would need to validate the removal of the first antibody before adding the second. It's far better to put the second Ab on fresh carboxylated gold.
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Hi dear colleagues
I have some problem at step of Aptamer attachment to plga NP with size of 250 nm. We use common EDC/NHS protocol of related articles. Protocol: 5 mg/ml Np+ 200mM EDC+100 mM NHS (MES buffer, PH: 5.58)-----> stirrer at room temperature(15-18 C) for 1 hour ------> 3 times washes -----> diapers pellet of activated Np by Aptamer suspension in 1 ml phosphate buffer(pH:7.5 - 8)------> stirrer at room tem for overnight
1. After agarose gel electrophoresis (2%), we didnt observe efficient attachment. what could be wrong in our protocol ???
2. Can use agarose gel by lowered percentage like 0.8 for differentiate of NP and NP-aptamer ? because both of them in 2 % agarose gel were placed in the wells and is so difficult to differentiate them .
3. Do we have another methods for confirmation of aptamer attachment to PLGA Np except of XPS? because we do not access it.
4. How about UV spectroscopy ?
So thanks
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Thanks for the suggestions.
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Hello
I am using EDC-NHS reaction to activate 4-carboxyphenylboronic acid (COOH-PBA), then coupleing with NH2-PEG-SH (Mw 2000).
What I want to achieve is the coupling between -COOH of COOH-PBA and NH2 of NH2-PEG-SH, while the -SH is remained. The final product is PBA-PEG-SH, which is used to functionalize silver nanoparticles via Ag-S bond.
The protocols is as following with some modifications from a paper 1:
Step 1: 100 ul 10-3 M EDC aqueous solution and 40 ul 10-3 M NHS aqueous solution is added into 1.5ml centrifuge tube;
Step 2: 200 ul 10-4 M 4-carboxyphenylboronic acid aqueous solution is added into above solution, under mild agitation for 45 min in darkness, to activate -COOH;
Step 3: 167ul 100uM NH2-PEG-SH aqueous solution is added into above solution; under mild agitation for 24 h in darkness to finish the coupling between -COOH and -NH2;
I am not sure if the conditions are ok for achieving my purpose. I would appreciate it a lot if you can give some instructions/suggestions/papers.
Thanks a lot.
  1. Verdin, A., Malherbe, C., Müller, W. H., Bertrand, V., & Eppe, G. (2020). Multiplex micro-SERS imaging of cancer-related markers in cells and tissues using poly (allylamine)-coated Au@ Ag nanoprobes. Analytical and Bioanalytical Chemistry, 412(28), 7739-7755.
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From my point of view, the protocol is suboptimal.
It might be preferable first to attach the NH2-PEG-SH to the nanoparticles and remove any excess if possible. To activate a carboxylic acid, I would start with a solution of the acid (often MES buffer is used), then add NHS, and finally the EDC. Incubation for e.g. 30 min should suffice. The nanoparticle solution should have a pH of 7-9 (no amines, borate buffer). The NHS ester would be added to this solution. A final wash/separation of the particles from the reagents would be the last step.
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Dear all,
I'm working on coating gold nanoparticles (AuNP) via EDC/NHS coupling and blocking them. And I came across a few hurdles that need your help.
1) Is there any precuations when functionalizing AuNPs and proteins with EDC/NHS respectively? e.g. pH, Temp., time, buffer, etc.
2) The AuNPs are reduced and stabilized by citrate and hence have (-) charge. I reckon common blocking agents like BSA, HSA, Skim milk have (-) charge at pH 7. I might have to perform the protocol at considerably low pH because those blocking agents have (+) charge below pH 4~5. I'm concerned that it might affect the colloidal stability or AuNP-protein interaction.
Would there be any way that I could block AuNP@Streptavidin without this wide range of pH change?
Thank you for your help.
Andrew
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EDC/NHS renders the Au nanoparticles unstable if they are coated with citrate ions. EDC/sulfo-NHS is supposed to be better at this, but I have never corroborated this. When adding the protein to the nanoparticles (considering that they survived the activation process), physical adsorption will most likely dominate over chemical linkage (moreover NHS is extremely susceptible to hydrolysis). I recommend running physical adsorption with streptavidin up to the point where the nanosystem is stable. Afterward, you can check blocking agents, again physically adsorbed. If the protein is expensive, I would first stabilize the nanoparticles using thiol-peg-cooh or thiol-peg-amine, I prefer the latter.
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Does addition of NH2 group at 5' or 3' end of aptamer has any role?
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Thank you
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In my process I want to conjugate ascorbic acid with chitosan
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Dear Rana Kamal, you can use only EDC and it will work for carboxyl group activation. Although, keep in mind that in ascorbic acid you don't have carboxylic groups .
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I have been trying to attach antibodies to glass for a while and can't seem to figure out the flaw in my methodology.
After adding amine groups to the surface using 2% aminosilane (this works perfectly as my contact angle measurement shows it). I use the protocol below
Protein #1: 10ul of antibody to 1ml of MES
Protein #2: 10ul of antibody to 1ml of PBS
1. 0.4mg of EDC (final concentration 2mM) directly to 1mL of Protein #1
2. 0.6mg of NHS to the reaction
3. Mix reaction components well and react with surface for 15 minutes at room temperature.
4.  Add protein #2 and incubate for 2 hours
5. Add the secondary antibody for detection (HRP) and incubate for an hour
6. Place surface in PBS and visualize under a microscope
Is there something I am missing or doing wrong? All washing steps have been removed for simplicity.
Thanks in advance for your inputs
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Hi Maryam,
You need the activated carboxylic acid group to bind antibodies. Your protocol does not seem to include it.
I have copy-pasted the protocol which I used to bind antibodies to silver nanostructures on silicon. Since the chemistry is similar, it must work on glass too.
For functionalization, the substrate was first treated in oxygen plasma chamber, maintained at ~400 mTorrand 10W for 5 min. Next, it was treated with 50 µL of 2% solution of 3-aminopropyltriethoxysilane (APTES) prepared in ethanol for 90 min. After cleaning with excess ethanol to remove unreacted APTES, the substrate was treated with 50 µL of 1:9 mixture of 20 mM 11-Mercapto undecanoic acid (MUA) and 3-Mercapto propionic acid (MPA) for 18 h at room temperature. This step generates the carboxylic acid group, required for molecular binding, on the metallized substrate. Next, the substrate was washed with ethanol to remove excess acid and treated with 50 µL of 1:1 mixture of 0.15M N-(3-dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride (EDC) and 0.03M N-hydroxy succinimide (NHS) for 1 h at room temperature, to activate the carboxylic acid groups created in the previous step. Finally, the substrate was washed with phosphate buffer saline (PBS) to remove traces of EDC and NHS. The reactive –COOH group thus obtained allows binding proteins and antibodies for biosensing applications.
Hope this information helps.
Best wishes,
Pearlson.
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Apologises I'm really confused and don't know how to do, appreciate any guidance you can give
Aim
1. To explore if anxiety is predicted by stress and treatment delay and whether this is moderated by Brief Cope strategies (Brief COPE) .
Design
1 continuous outcome variable – anxiety (let’s call this H)
2 continuous predictor variables (let’s call these D, S)
3 Continous Moderator - (lets call these BC - ef, bc pf and bc avoidant). These are inputted into SPSS as 3 separate variables as the questionnaire b-cope does NOT allow you to create a total score (by adding ef + pf + avoidant).
- D – delay
- S – Stress (measured by pss-10)
- BC pf - Brief Cope - 1 (consists of 4 questions with each questions represent a different factor)
- BC ef - Brief Cope – 2 (consists of 9 questions with each questions represent a different factor)
- BC avoidant - Brief Cope – 3 (consists of 4 questions which each questions represent a different factor)
To answer the aim I know i need to complete a hierarchial multiple regression but I don't know what to enter on what model or whether I need to do separate regressions and again what should be entered with what.
Q1. Can you please advise how my regression models would look as I can't work this out given my predictors, moderators and outcome variable listed below.
E.g. Model 1 ...
Model 2 ...
Q2. Do I need to look at interactions? If so which ones, how would this be put into SPSS ie in which models.
Possible Interaction examples ?
Stress x bc ef
Stress x bc pf
Stress x avoidant
Delay x bc ef
Delay x bc pf
Delay x avoidant
Q3. Do I need to run separate hierachial multiple regressions? If so can you please write how the model would look ie. Model 1 ..
Model 2...
To confirm I have completed only parametric tests. I have 1 group completing all predictor /moderators variables.
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It sounds like you would end up with a regression model in which you have a very large number of predictor (independent) variables as well as many interaction terms (since you have so many different COPE variables). My advice would be to first think about meaningful ways to reduce the number of COPE variables to be included in the model, for example, by aggregation (calculation of a summary COPE score), factor analysis, or simply selection of the theoretically most meaningful COPE variables. Otherwise you might run into various problems when entering all individual COPE items into the regression (e.g., potential collinearity, too many individual significance tests, large model with many predictors, loss of power to detect interaction and other effects).
Other than that, you could run a hierarchical regression model with only the main effects (predictors, no interaction terms) in the first model, then add the interaction terms in Model 2 to see if they add anything to the prediction of the outcome. But, once again, I would try to reduce the overall number of predictors first to avoid problems in the analysis.
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I use EDC/NHC to activate PEG-COOH so that aminated GQDs can be modified
But the result is often not decorated
And I can't find where the problem is
The following are my experimental steps
PEG was dissolved in deionized water then 0.1M MES PH6 was added
Dissolve EDC/NHS in MES and add the solution to react overnight
PEG/EDC/NHS molar ratios are both 1:1:1 or 1:2:2
There will even be a small amount of white line-like objects in the reacted solution.
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You could add NHS first and dissolve EDC (better use EDC-HCl, it's more stable) in DMSO. Then react with PEG-COOH at RT for 15 to 30mins, then initiate the coupling at slightly alkaline pH.
Find a way to remove excess EDC.
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I am looking to create a SAM layer on a 20 x 20 mm gold surface using EDC and NHS which will link to Igg antibodies. However, I want to know if there is a way of calculating the exact/correct amount of linker solution needed for the surface are?
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Nearly all reactions in this field are performed with a large excess of reagents. By washing your gold surface, all unreacted reagents can be removed easily. Please note that IgG molecules are much larger than the linker molecules and that you do not need to use all linker molecules for the conjugation. In addition, it might be helpful to use an oriented way of antibody conjugation, e.g., by Protein A or G.
Finally, it should be mentioned that you won't end up with an IgG SAM anyway.
BTW: Many proteins can be firmly adsorbed on plain gold without any linker. I would try this first.
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I have COOH functionalised nanoparticles and would like to conjugate Temozolomide(TMZ) to these nanoparticles. I wonder about EDC/NHS coupling will work as TMZ has NH2 groups in it.
To maintain the stability I would perform the experiment at a pH of 3(MES buffer). I appreciate some suggestions
Thanks
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I don’t think this will work. Your ‘amine‘ is actually an amide and has greatly reduced reactivity because of the carbonyl group. A separate point is that you cannot use MES at pH 3, at least not as a buffer, as its pKa value is 3 orders of magnitude higher, hence it has no buffering capacity at pH3
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In most cases, EDC/NHS coupling for carboxyl group activation of carbohydrate or protein generally accompany the acidic pH (5-6). but i found unusal paper which adjusted pH to 9 for carboxymethylcellulose amination with alkyl or alkylene diamine in Carbohydrate Polymer.
Then, what is the accurate interpretation of pH adjustment in EDC/NHS coupling?
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In your original question you mentioned diamines, but for thiolation you need to take a different approach, as thiols react with EDC, and also crosslink as you say. Is cysteine important or do you just need a thiol? A better approach I think is to use cystamine (note NOT cysteamine), which is a diamine with a reducible disulfide bond. You can therefore make a stable derivative for storage and reduce a portion with DTT as and when needed, followed by desalting to remove excess thiols.
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i am asking about conjugate monoclonal antibody to the surface of NH2 modified MNP i have 2 choices the first using NHS and EDC to activate the Ab first is it safe for antibody activity?
the second one is using glutaraldehyde as a crosslinker on the particles, is glutaraldehyde could react with NH2 in the Fab region ? so the antibody will loose the activity ?
which is better?
is there any precise source for the conjugation protocol?
then what about stability of functionalized particles is it required to use preservative and what is suggested preservative please
thanks :)
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Thanks for your separate message. As you definitely have to use amine beads, look up "two-step glutaraldehyde" which may be the best way forward.
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If thiol and amine groups are present in a polymer backbone,
how can selectively be carryout coupling of the amine with any carboxylic acid without disturbing the thiol group?
Will the EDC/NHS reaction be useful?
Finally I want free thiol group in the polymer chain after coupling of the amine with carboxylic acid..
Please suggest me, if any useful literature is available...
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Amine is more reactive than thiol in EDC -NHS coupling reaction. Please refer the following paper.
pH- and GSH-Sensitive Hyaluronic Acid-MP Conjugate Micelles for Intracellular Delivery of Doxorubicin to Colon Cancer Cells and Cancer Stem Cells
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I am trying to graft folic acid on PHA with 0.5 mmol NHS and EDC.HCl 0.5 mmol. I used the solvent DMF. but my product is very low. i got only 15 mg polymer from 50 mg initial weight. Is there any suggestion on how to increase yield? With the same concentration previously i got high yield with chitosan using acetic acid.
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You wil want to check all your synthesis and purification procedures. 1) you have to ensure that all chitosan is dissolved before initiating the reaction, also ensure that you have activated the carboxylic group fully before introducing it to the amine group, you can do this by reacting the EDC with the folic acid for an hour at least before introducing it to the fully dissolved chitosan solution. 2) you are likely to loose products depending on your purification techniques, I will suggest you try using dialysis for purification with a molecular cut off that allows for good separation. you can check
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Hi Everyone,
Our group recently is trying to bind ProteinA on the acid-functionalized electrode through the EDC/NHS chemistry. We have seen the change through the activation of EDC/NHS but the following ProteinA-binding step just brought the impedance back to the same level of the unactivated surface (By EDC/NHS). Similarly, the other set immersed into PBS buffer presented the same impedance change which we thought may be caused by the hydrolysis. However, the success of activation has been proved by attaching other redox-active molecules containing primary amine. Does anyone have any ideas about this phenomenon? We presumed that the PI value of ProteinA was the issue but no matter we tried the high PI protein, avidin, or lower the PH, like 4, the supposedly increased impedance was not observed throughout the experiment.
Experimental details:
We activated the acid in the MES buffer at PH=6 and implemented the binding reaction in PBS buffer at PH=7.4. Binding and activation time is constantly 2 hours but longer time was also tested and no difference was realized
Will appreciate it if anyone could help with this problem.
Thanks.
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Michael G. Weller Thanks for your time in answering this question. We used PBS buffer throughout all ProteinA binding trials. According to the vendor, there should be no primary amine in the formula. The PH for binding is 7.4 but we also tried 8, and 9. Still, no sign of binding was seen. In terms of EDC, we stored it in the freezer in the glove box and only took the amount we need for the experiment out right before the EDC/NHS treatment. We indeed used only 0.1 mg/ml for the conjugation trials and 1mg/ml should be a good try, but this could be the highest we could get according to the solubility of ProteinA (1 mg/ml). Thanks again for your valuable thoughts regarding my question.
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Hi
The protein that I try to conjugate with AuNPs has no cysteine, so I modified my AuNPs with COOH-PEG-SH groups. But the conjugation doesn`t proceed yet. I tried all recommended procedure but I didn`t get desirable results yet.
There is some probability for this observation:
1- the protein I work with may be deactivated after conjugation.
2- the carboxyl group of AuNPs may not been activated using EDC/NHS, however I try all recommended procedures including, MES buffer and deionized water in pH range between 5- 6.5
My question is:
May SH-PEG doesn`t have enough carboxyl group, because I ordered it from a laboratory to synthesize and attach it to AuNPs that I`ve synthesized before. How I can be sure that my PEGylated AuNPs are carboxyl terminated?
My second question is how I can be sure that the protein -AuNPs conjugation without using dot blot method? (My protein is recombinant , and its antibody is unavailable). Is SDS-PAGE applicable?
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Dear Samaneh Mirsian thanks for sharing this very interesting technical question with the RG community. There are various helpful literature references about this topic available. For example, please have a look at the following relevant paper:
Conjugation of Polymer-Coated Gold Nanoparticles with Antibodies—Synthesis and Characterization
The good thing about this paper is that it is freely available as public full text on RG, so that you can download it as pdf file. please note that this is just one typical article in this field. You can find and access many more by searching the "Publications" section of RG.
It might also be helpful reading the answers given to some closely related questions which have been asked earlier on RG. For example, please check the following threads:
Conjugation of PEG to AuNPs?
(14 answers)
and
What are the techniques to conjugate antibodies to PEGylated gold nanoparticles?
(4 answers)
Good luck with your work and best wishes, Frank Edelmann
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Will there be any visible changes in the colour and viscosity
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Thank you so much for your valuable reply Sir
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Hi all,
I am trying to conjugate an NHS ester to a solution of gelatin in PBS. So far, I am only finding protocols on conjugation of the dye to antibodies, which is not terribly helpful since they all say to avoid having gelatin present in your aB samples. Has anyone successfully conjugated a dye to gelatin? The dye in question is the Alexa Fluor 405 NHS Ester.
Thanks!
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All standard protocols should work. Usually, antibodies are to be labeled and not gelatin. This is the reason why gelatin (and other proteins and amine-containing compounds) should be avoided. Suitable buffers are borate, carbonate, or phosphate at a pH of around 8.
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I want to conjugate APTES with COOH-PEG-Folate. The purpose is to make amide bonding between -NH2 of APTES with COOH of COOH-PEG-Folate.
Is this possible by EDC/NHS crosslinking? Which medium is suitable DMF/DMSO or PBS?
I would appreciate your suggestion and also if someone can share protocol on it if you are familiar with.
Thank you.
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Dear Riaz A. Khan Yes there is free -COOH available in COOH-PEG-Folate.
Thank you.
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I have functionalized 20 nm AuNPs with SH-PEG5k-COOH and SH-mPEG5k in a 9 :1 ratio. The final solution is around ~1 OD in DI water. To react with EDC/Sulfo NHS, I am using the following conditions.
MES buffer at pH 5.4, 50 mM, 5 mL
AuNP@PEG-COOH/mPEG OD ~1, 1mL in MES buffer
EDC/NHS ~ 10 ug each (stock solutions are prepared in DI water), 100 uLwas added soon after preparing
Reaction time- tried both 15 min, 30 min
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Thank you, Nick Gee and Ahmed Jehanli
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Dear all,
In synthesis process of curcumin loaded albumin nanopaticles, when I used EDC as crosslinker, surprisingly it caused intense aggregation! Actually I used these methods :
Does anyone have any experience what the problem is? Could the problem be the ratio of albumin to EDC? In this case, what is the best ratio?
Thank you.
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I don't think it's surprising! As you have a protein with multiple amine and carboxyl groups, you simply have too much EDC and are causing excessive crosslinking. There is no 'best' amount of EDC that can be applied in all applications. The 'correct' amount will be determined by concentration of the BSA and other reactants, buffer type, pH, temperature, time of reaction, quality of the EDC etc. I think you just need to titrate the amount of EDC, and clearly the optimum amount will be below the level you used last time.
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Hello everyone,
My research will interview people living with dementia outside of NHS settings. In the University ethics application I outlined the use of a consultee to ensure people living with dementia who lack capacity have the opportunity to participate. However, what the NHS HRA defines as ‘intrusive research’ involving adults lacking mental capacity cannot be approved under the University's ethics processes, as University ethics committees are not recognised as Appropriate Bodies under the Mental Capacity Act . This is regardless of whether the research is taking place within or outside of the NHS.
The only option I can see to avoid HRA application, is not including people who lack capacity to consent. I do not want to take this route as it seems inequitable. This doesn't sit well with me and I wonder if anyone has confronted this issue in the past?
Any advice and information welcomed!
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In order for adults lacking capacity to participate in non-clinical trial research it must be connected with an impairing condition in the functioning of the mind or brain affecting the person (or its treatment), which causes or contributes to the impairment. a person must be assumed to have capacity unless established otherwise. Individuals should be helped to make their own decisions as far as practicable. A person is not to be treated as unable to make a decision merely because he makes an unwise decision. All decisions and actions must be in the best interests of the person lacking capacity. All decisions and actions must be the least restrictive of the person’s rights and freedom of action. Capacity refers to the everyday ability that individuals possess to make decisions or to take actions that affect them, from simple decisions such as what to have for breakfast to far-reaching decisions about serious medical treatment or financial affairs.
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I am having a hard time trying to figure out the mechanism of 4-aminothiophenol going through the EDC/NHS coupling reaction. Where the 4-aminothiophenol that is linked to a gold nanocage will be crosslinked to a protein either in the c terminus or n terminus.
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From the wording of your Q, it seems like the thiol will be interacting with the gold surface prior to conjugation? This may direct the EDC reaction more towards the amine, but you'll just have to try I guess. The 4-aminothiophenol doesn't give you very much 'spacing', but it may be okay depending on the application.
I'm not sure how you will get directed attachment at either the N-terminus or C-terminus with this chemistry, as the amine will react with carboxyls which are likely to be found throughout the protein.
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Hello everyone,
Can anyone please tell me, how can I couple polymeric alcohol to phenolic acid?
Some forums suggested two kinds of reaction, first by mixing it in presence of H2O2 and EDC & NHS coupling. These two reactions work for me, I got FTIR and NMR results from both of the reactions and good yield but I couldn't find the exact mechanism of the reaction.
Thank you for your help in advance.
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Dear Experts,
I am a beginner in Lateral Flow Assay. I am doing the conjugation. It appears that there are many kinds of gold nanoparticles to choose: bare particles, Carboxyl coated or NHS coated particles. I do not know which kind should I start with. As far as I know, the bare gold nanoparticle is a traditional one and the protocol is not difficult to do but the attachment maybe dissociate.
Thank you for your kind help!
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Omar Gonzalez-Ortega Gracias Professor. It's very kind of you to answer all the questions. I really appreciate your help. I have some more questions but I should explore by my self first.
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Hi there, I am doing functionalization of a citrate based polymer with EDEA using EDC Chemistry with NHS and MES. Can someone help me find a protocol for using EDC on a gel scaffolds. Most of the protocol I can find is by dissolving their protein or nanoparticle in MES buffer before adding the EDC and NHS. My sample is a gel and not in particle or in powder form and I am having trouble on how much and of what concentration of these reagents can I use to soak my gel and later on conjugating my amino. Hope someone who’s doing related research can help me with this one. Thanks
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Hello,
We found a protocol online to generate CM chip.
Wash it by water X 3, dry it by N2 gas. Spreaded 70 μL of 20 mM cystamine hydrochloride onto the Au film, put into a box, overnight at room temperature. Wash it by water X 3, dry it by N2 gas. Then, the 70 μL mixture solution of 15 mM EDC, 75 mM NHS, and 10 mg mL−1 CD was dropped onto the cystamine modified Au surface for 2.5 h and the Au surface was cleaned by water X 3, dry it by N2 gas, and stored at -20C.
The coupling efficiency is not good and it's difficult to find another protocol using EDC/NHS to coat dextran. Can you pls give a suggestion?
Thanks.
Shawn
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to solve this problem, we ordered three batches of EDC/NHS from different companies, but none works.
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The military, both serving and veterans and their spouses and families, can have needs that may be different from civilian patients. There is a covenant between the NHS and Armed Forces. Are healthcare staff aware of the covenant?
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If the gap between medical resources is widened, I think it is an issue that should be paid attention to.
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We customise 30um NHS decorated PS/DVB bead from EPRUI and want to immobolise streptavidin onto it. Yet verifying by biotin-FAM probe, the immobolisation is mostly negative.
Could you please provide some advice among our protocol and method?
Our protocol is as below:
0. The bead is transported and kept in isopropanol 2-8℃
1. To use, the bead was washed with 4℃ 1mM HCL for 15s, then centrifuge and discard supernatant
2. add equal volume (100ul bead~100ul protein solution) of streptavidin (pI=6.0) solution (3mg/ml in pH4.8 0.1M MES buffer)
3. Room temperature incubate for 2 hour
4. block with Tris-EDTA buffer for 2 hour
5. centrifuge, discard supernatant, wash with Tris buffer again then add 1ml 1nm/ml biotin-FAM probe in tris buffer
Thanks!
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Thank you for the kind support Michael G. Weller Steingrimur Stefansson . We stepped back to carboxyl bead and it works, ridding off the transportation problem. We are also considering bead functioned with epoxy group, which are also not so sensitive to temperature
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I'm in engineering, so have less perspective on some biological procedures. I've been immobilizing antibodies onto sensors for a project, and I'm not sure how to properly dispose of the waste. Can I mix everything together or do I need to dispose of the waste from each step separately? I have: 11-mercaptoundecanoic acid in ethanol, MES solution, EDC/NHS solution, antibodies in PBS, 1M ethanolamine, and 1uM BSA solution. I have a small autoclave, ethanol, and bleach in my lab, and chemical waste disposal pickup available.
Thank you in advance!
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Thank you!
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I have a project related to antibody conjugation and I will use NHS/EDC. Can I reuse NHS/EDC? Thank you!
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Hi Chun Wai Wong . Sorry, no. The reagents are single use once they are dissolved.
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Amine Biotinylation Reagents (especially NHS-biotin) reacts with primary NH2 group, while A, C,G base all have NH2 group. So I wonder if these Amine Biotinylation Reagents would also biotinylation nucleic acid (DNA and RNA) when treating cell lysates.
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Hello, I have developed a Surface Plasmon resonance sensor using LED of wavelength 635nm and CMOS webcam as source. I am using the diverging rays of the LED as the change in incident angle. When I put silver coated glass slide on the prism I get a dip at a particular angle. I have test the sensor by immobilizing with MUA , EDC/NHS and IgG. The sensor can detect the shift in angle for all the layers. But when I put liquid dielectric medium like DI water, BSA or PBS buffer the shift disappears. I can monitor real-time data with the webcam and so when the liquid sample is passed I should be able to detect the shift. I have attached the file of how the dip looks like.
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Hello,
For in vivo experiments, I am coupling Cy5 to linear peptide hydrogelators at the N-terminus. I could successfully couple Cy5 (mixed with DMF) to the hexapeptide (H-Phe-Gln-Phe-Gln-Phe-Lys-NH2) by SPPS (coupling time 4h), but I cannot couple it to the extended decamer sequence (H-FQFQFKFQFQFK-NH2) even after coupling it for 4h or overnight.
Can someone tell me what is the issue?
Thank you in advance
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The amide coupling yield is strongly affected by pH. You should try with a tertiary amine as a catalyst.
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Hello everybody,
I tried to centrifugate the COOH-microspheres (polystyrene) with a diameter of 7 μm after adding EDC and NHS dissolved in 0.1M pH 6.0 MES-buffer (final concentration: EDC 40mM and NHS 5mM) at 9600 x g for 5 minutes at room temperature. But they could not form a pellet and the solution looked turbid.
Later, I added ethanolamine solution (100mM) to them and tried to centrifugate them again, but it still didn’t work.
In the beginning, I washed the microspheres with 0.1M pH 6.0 MES Buffer, and they had no problem forming a pellet at the same centrifugation conditions. I don’t know why it didn’t work anymore after EDC and NHS being added.
I will be very glad if anyone could help me with this.
Thank you very much in advance.
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It is common problem.
Take a look this protacil it is the best one if tou work with polyester.
Use the conditions which written here.
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I want to start a discussion on why bioconjugation scientists choose NHS, TFP, or PFP esters for conjugating to amines. TFP and PFP esters are less susceptible to spontaneous hydrolysis in aqueous solutions than NHS esters. However, they are somewhat more hydrophobic, and their optimal pH for conjugation is slightly higher than the optimal pH for using NHS esters in bioconjugations.
If you use NHS, TFP, or PFP esters for bioconjugation to amines, what are your reasons for choosing one type of ester over another? I want to hear as many reasons for doing so as I can obtain. If you have published references that help support your reasoning, I'd love to see those.
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Hishar Hassan thanks for the question!
The reason that DMF and DMSO are so frequently used to dissolve NHS esters is that they can be made anhydrous (for example, by drying over 3 Å molecular sieves), and they are miscible in water. When a compound containing an NHS ester is dissolved in water, it starts to hydrolyze immediately, and the half-life of the ester depends on the pH. At or below pH 7, an NHS ester in water has a half-life measured in hours, but as the pH rises, the half-life gets progressively shorter. At pH 8, an NHS ester has a half-life measured in minutes. The short half-life of NHS esters in water is one reason why people look for alternative active esters such as TFP and PFP.
DMF is a bit tricky to use since it can degrade over time, forming free amines that react with the NHS ester. You should always use fresh DMF of the highest grade possible, and you should dry it over activated molecular sieves for >=48 hours before using it to dissolve your NHS ester. A good alternative to DMF is N,N'-dimethylacetamide (DMA or DMAC). It has similar properties to DMF, but it doesn't degrade over time to form free amines.
I don't prefer to use DMSO. In my experience, DMSO takes longer to dry than DMF or DMAC, and it is harder to get rid of than DMF or DMSO.
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Premise:
Standard surface modification for biosensing involving NHS/EDC amine coupling to a COOH-SAM (functionalized AuNPs), but instead of ethanolamine that is normally used, Tris-Cl (pH 8.5, 2M or 1M) is used to block excess activated NHS groups.
Both should result in some amount of COOH to OH conversion, with Tris having more.
Expectation: no relationship to HIS tags
Reality: Covalent strength (not removable with EDTA, HCl, imidazole or anything else viable on a biosensor) coupling of HIS tagged ligands.
Has anyone experienced this in any situation?
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can explain more?Please
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I am trying to connect the antibody using DSPE-PEG-COOH, but it seems to be unsuccessful. The experimental steps are as follows:Take 1mL liposomes into a 1.5mL centrifuge tube, wash once with 1mL PBS, and remove the supernatant after centrifugation at 16000rpm at 4℃ for 10min; add 400μL EDC (5 mg/mL) and 400μL NHS (5 mg/mL) respectively The solution was added to the centrifuge tube, mixed thoroughly, and then placed in a 37°C water bath to activate the carboxyl group for 30 minutes. Then 1M NaOH was added to adjust the pH to 7.4. Add 25 μL(100μg/mL) of PCT antibody and react at room temperature for 6h. After washing twice with PBS, resuspended in PBS (add 1% BSA) to obtain liposome@Ab2 and store it at 4℃ in the dark for use. But in the follow-up, the ELISA experiment was not successful. Is the concentration of the antibody I added too low? or other reasons?
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Thank you so much, I will use your suggestions to adjust my experimental steps as soon as possible, which is very helpful to me. At the same time, the traditional ELISA experiment will be tested without liposomes to make sure that there are no problems with my other experimental procedures. If the experiment is successful, I will be the first to tell you. @Michael G. Weller
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Hello everyone!
I am conducting a systematic review and CRD (NHS EED and HTA) is one of the databases I am searching. I wanted to export the articles and import the to a reference sofware but it is only in a tagged text format and not supported by by the ref software.
So has any one encountered such a situation and if you have some suggestions to handle it?
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OK, for future reference, I worked it out. After downloading the filter from the CRD website (guide to searching) and adding it to the import filters (I don't know if this was just by opening it, or by copying it into the EndNote Filters folder on my C Drive - I did both). Then, rather than import the text file using the CRD filter, I just had to drag the text file into my EndNote window, confirm the CRD filter and it worked.
Yay.
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Hi there,
I am trying to measure dendritic spines from mouse fixed tissue, specifically, I am looking at the prefrontal cortex. I injected the PrL with a lentivirus to knock-down my protein of interest and now I would like to measure any differences in spines. I can't use Golgi staining because it will get rid of my transduction. I tried IHC but all I see is fluorescent cell bodies and not dendrites. Has anyone tried IHC to measure dendritic spines in the PrL? If so, is there a way to improve the IHC so the dendrites are visible?
Mouse was perfused with PBS and 4% PFA
Brains cryoprotected with 30% sucrose
Frozen Sections at 20 um.
My protocol for IHC is
10% NHS blocking soln
Anti-GFP(1:3000) in 1% NHS blocking soln overnight at 4 degrees
Wash 3x15 mins
Alexa-Fluor 488
Imaged at 60X on the confocal.
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Dear Nadine,
Any chance you could change your GFP reporter to membrane-targeted version. This would likely homogenize the GFP signal trough the entire neuron including dendrites and spines. You could also bleach your samples in peroxide solutions to reduce background before GFP labeling. Tissue clearing protocols may also be a strategy to consider.
Cheers,
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Hi all, I am preparing antibody functionalized carbon nanotube in suspension through EDC/NHS chemistry. Briefly, I am activating the -COOH group attached to my SWNT by EDC/NHS in MES buffer and I am starting to see agglomeration of the CNT in the solution. The CNTs are dispersed but in bundled/agglomerated form. After centrifugation and washing and subsequent addition of antibody, I am experiencing slightly larger agglomeration of the CNT, which if kept at rest, settles down. I haven't used any surfactant (like tween 20 or Triton-x) yet. Is this normal to observe this kind of situation while functionalizing CNT in solution/ suspension? Thanks.
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Sulfo-nhs instead of nhs might help
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Ester formation from COOH (Carboxylic acid ) and OH(alcohol) by using EDC/ NHS is possible؟
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It surely uses EDC/NHS for coupling of COOH and OH. However, I prefer using DCC/DMAP for this coupling.
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Hi there!
I am working with hydrogels crosslinked with EDC, NHS and AAD and will like to know a way of keeping track of the reagents leaking out after each wash. So far I am thinking of taking samples of the washing solvent (water) and checking the amount of solids dissolved by evaporating and weighting the sample. However, it is a pretty rudimentary method and maybe you know something more specific.
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Hi Payal Bhatnagarr,
Currently, i am working on a PVA-GELATIN system which was chemically crosslinked by Glutaraldehyde
The washing step turned out to be a major saving to my investigation
I optimized using;
Tris HCl (3 M), PBS (0.1 M), ammonium chloride (50 mM) and distilled water and used the Benedict test as a colour indicative confirmatory test to know whether the excess -CHO was washed out.
Similarly, you should know your system well and choose appropriate washing buffer as well as the test to confirm washing.
The knowledge about the confirmatory test will help you optimize the washing time
Washing and optimization is a common phenomenon;
Hope it helps
Regards,
Benu
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Looking to label a enzyme (at it's -COOH groups) with an dye with an amine or cadaverine group. (I can't use an NHS ester here, two important sites of the enzyme would be compromised). There are lots of dyes available, but I'm surprised that I can't find a manual/protocol out there.
Basic gist is to mix protein with a large molar excess of dye (100:1) then start reaction by spiking EDC, stopping with Zeba column purification. Protein cross-linking is obviously a concern, hoping the large molar excess and a shorter reaction time help mitigate this problem.
Any advice/experience on buffer pH, ionic strength, EDC concentration, etc. for this reaction?
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Hi Matt. I was wondering if you could reach a final protocol for this question. Which EDC concentration did you use? Which protein concentration? Thanks!
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Hi!
We are aiming to bind streptavidin to polyacrylamide surface. The EDC/NHS method bounced on me as a common method, utilizing the -NH2 of polyacrylamide and -COOH of streptavidin. Is there any unconsidered factors that may block this route? If EDC/NHS is viable to immoblise streptavidin to acrylamide, how should the reaction condition be set? Consider the static charge of polyacrylamide and streptavidin ( isoelectric point = 6), which pH is optimistic? Should we use a 2 step EDC/NHS xlk or 1 step?
Thanks!
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You may add 5 % of acrylic acid to your acrylamide. Subsequently, the polymer can be activated by a carbodiimide/NHS mixture.
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An impurity formation observed up on storage of mPEG DBCO. Can any one suggest additives to make it stable...also best reaction conditions for preparation of DBCO mPEG from DBCO NHS and mPEG amine
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Store at -20°C; Desiccate; Avoid frequent thaw and freeze.
addition of a slight excess of (MeCN)4CuBF4 quantitatively formed an isolable octyne–Cu complex that is unreactive towards free azides. The Cu–alkyne complex could be disrupted by the addition of aqueous ammonia, regenerating the cyclooctyne and making it available for strain-promoted azide–alkyne cycloaddition.
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Hi,
I am trying to use EDC/NHS to attach collagen to PLLA and coming across issues so would be grateful of any input!
My protocol is:
(1) Aminolysis the PLLA surfaces for NH2 group exposure
(2) EDC/NHS (100mM) in MES buffer at pH 6
(3) Addition of collagen (in 0.2% acetic acid) to the EDC/NHS buffer and stir for 1 hr
(4) Addition of the PLLA material to this solution and incubation for 3 hrs
(5) Rinse polymer and dry
Chemical functionalization isn't an area I have a lot of experience in so any help would be great.
Thanks!!
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You should not use acetic acid or any other carboxylic acid in the activation step. It reacts with EDC and the conjugation will fail.
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I'm conducting experiments using carboxyl-amine coupling via high concentrated EDC/NHS reagents in aqueous condition(MES buffer & PBS). After few minutes, some bubbles(gas?) were generated in the bottle. I were trying to check which elements can produce bubbles, but I couldn't find the solution. Could you tell me why there were some bubbles during EDC/NHS coupling?
Thank you!
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Mohamed Taieb Bakouche Thank you for your comment! I will check about the presence of ethyl isocyanate in my reaction mixture.
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Has the presentation given at Symposium - Child Protection Special Interest Group: Child protection and safeguarding training - Is simulation training effective? by A Thomson, P Nayak, M Plunkett and C Kallappa been written up?
The Abstract appears as G170(P) in Archives of Disease in Childhood, vol 99, issue suppl 1
I work in the library of an NHS Foundation Trust and one of my colleagues, a
Mental Health and Learning Disability Education Team Lead, would like to learn more for a project she's working on
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هو جزء من منظومة تعليمية مكملة للتعليم بالتاكيد يساعد
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How to correctly covalently attach a protein through NHC / EDC if pI 10?
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Just avoid pI when you are choosing buffer for reaction
My best regards, Antonina
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Hi,
I am planning on running some protein-protein interaction/kinetics studies using the Nicoya COOH sensors in OpenSPR. I am looking for the activation and blocking buffer composition that are needed for this coupling (EDC/NHS). Anyone here has experience using these sensors from Nicoya? Could you share your detailed protocol and buffers (composition?
-Thanks
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Amine Coupling: A ligand is immobilized onto slide with available carboxylate groups. The carboxylate groups on the surface of the slide are modified with linker, a mixture of EDC and NHS to form an ester. A covalent amide bond is then formed to the ligand which must have at least one free primary amine group. Injection of ethanolamine removes any loosely bound ligand. Analyte is injected over the surface at a range of concentrations higher and lower than the expected equilibrium dissociation constant (K).
Chemicals:
EDC (Sigma Aldrich Cat. No. 03449-5G)
NHS (Pierce Cat No. 24500) 10mM
Sodium acetate pH 5.2 (need pH about 2 units below pI of ligand to be coupled)
1M ethanolamine pH 8.5 (Sigma >98% Cat No. EO135 – you dilute to 1M and pH to 8.5 with HCl)
PBST (Sigma Cat No. P 3563) or whatever buffer is needed
Immobilisation:
Flowrate: 20-50 µL/min
SPR prism temperature: 25 °C 4.
Steps:
Weigh 40 mg EDC and 10 mg NHS into a suitable vial and dilute with 0.9 mL of water. Dissolve and inject as quickly as possible after diluting. Inject over both channels for 7 minutes 20 µL/min.
Prepare a solution of 30-50 µg/mL of proteine in 10mM sodium acetate pH 5.2 buffer. Over the sample channel only, inject for about 10 minutes or until a maximum has been achieved.
Inject over both channels 1M ethanolamine HCl for 10 minutes (blocked the unspecific binding sites).
Inject the solution of the ligand (other protein) for both channel, than remove the excess amount by running buffer.
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I am working on the conjugation of antibodies on gold nanoparticles surface recently and I was looking for an optimized method for this purpose based on EDC/NHS. Given the many methods available, I would be grateful if anyone could suggest an optimized protocol with high efficiency.
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EDC/NHS chemistry is not very reliable and not an obvious method to conjugate antibodies to plain gold nanoparticles. On fresh gold particles, antibodies might be adsorbed directly or via a thiol group either obtained by partial reduction or a thiolation reagent.
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Hello,
my question is: why I get the best yield at pH 2.5 when I couple my antibodies to a NHS plate ?
I always thought that the optimal pH for amine NHS reaction is a basic pH.
Does anyone has a suggestion for my results?
Thank you in advance,
Ilja
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But I think thiol and hydroxy groups can still react.
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Which coupling protocol can be applied to get high yield for coupling, for example DCC, EDCI, NHS etc? Furthermore, how to purify and characterize the adduct? The concentration of the antibody is 1 ug/uL.
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This kit helps link Antibody and DNA based drugs
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I have been trying to conjugate folic acid with a peptide for a month now. i have used both EDC/NHS and DMF/DIC method but still there is no conjugation. On HPLC the product is coming at 2-3 R.T. My peptide R.T is 17 so now the conjugated product should be more hydrophobic considering folic acid is hydrophobic. I have no idea what it is but it is showing peak for both 370 and 254. Anyone please?
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Protocol followed as-
folic acid mixed in DMF , followed by addition of oxymapure and DIC, 2minutes incubation for activation then addition of peptide. Left for overnight on shaker.
Peptide is dipeptide and yes DMF is of conjugation grade, we use it for solid phase synthesis.
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I would like to label Human Serum Albumin for my experiments.
My supervisor suggested NHS and 6-Aminofluorescein as working agents.
Can anybody please suggest a working protocol?
Any suggestions on storing? Can I freeze the labelled protein in a solution?
If you have any other suggestions I would be very grateful!
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Obviously numerous examples of how you can perform the labelling can be found. It depends on whether you use the label(s) separately or as an already prepared ready-to-use sample like in:
In essence it comes down to the use of NHS in order to enable the fluorescein to get attached to your protein. Once the labelling is finished you need to rid of the excess of NHS and 6-aminofluorescein. In:
you see that they use dialysis to remove the excess of NHS and 6-aminofluorescein. In:
Bidmanova, S., Chaloupkova, R., Damborsky, J., & Prokop, Z. (2010). Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons. Analytical and bioanalytical chemistry, 398(5), 1891-1898.
the conjugates (CF–BSA) were separated from unreacted dye by size-exclusion chromatography.
Your question about storage. Indeed you can make one stock solution (let say 20 mL) and make smaller portions (let say 0.5 mL) and freeze them. Once you take a portion out of the freezer you better use it one time and better not freeze it back again (to be one safe side).
Best regards.
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I have been working with nanoparticles with different functional groups. Usually its well known protocol for conjugating cRGD or RGD with COOH functional group but I have a few particles which are amine functionalized, I found 2 articles but I am not yet convinced by different procedures they have used, can someone guide me with the process, if possible with experience and references.
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My research topic seeks to identify if the NHS leadership model is really used when redeploying nursing staff to support others areas (during sickness/staff shortages/emergency planning) in the practical setting of acute healthcare in the UK, and if so, which dimensions are utilised more than others.
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Good question.
In my experience, I have not seen the current NHS Leadership Model (or its predecessor, the LQF) used for making operational re-deployment decisions when faced with sickness, staff shortage or emergency planning problems.
That said, I have seen these frameworks used as part of both planned recruitment and leadership development activities.
Hope this helps
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Is the absence of decision by UK and NL to enforce social distanciation against Covid19 (unlike China, Italy, France, Spain, Germany, USA, etc) caused by memory loss of the positive effects of proactive enforcement of social distancing by US cities in the 1918 influenza pandemic? Death rates were reduced by 50%, source:
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Many thanks to all contributors. What emerges is that although the viruses of 1918 and 2019 are different, human behaviours in these two pandemics can be compared. Is it not notable that the portfolio of NPIs (non pharmaceutical interventions) is the same: handwash and reinforced hygiene, self-isolation, quarantine, lock-down, social distancing, gloves, face-masks?
100 years for not inventing better, and not learning from 1918 or from other countries (China/Wuhan) how to detect and implement early?
100 years to forget!
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Dear all,
I am looking for examples of feedback plattforms (crowd sourcing) that aim at the quality of service delivery.
Something like this: https://www.careopinion.org.uk/.
This platform collects feedback on the quality of the NHS in the UK and also displays reactions of the NHS. Do you know any other platforms?
Also, I would be very interested in research that analyzed the effects of those platforms on accountability and service delivery.
Thank you very much and best regards,
Thomas
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We have one platform in Brazil, which is very popular and is used by a large quantity of business: https://www.reclameaqui.com.br/.
Reclame Aqui Site is focused to show the level of trust of the enterprise to consumers, it also contains some business of delivery and provides some rankings based on the average rating, solution index, and repurchase.
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As we know, the immobilization of antibodies on the sensing platforms is the most critical step. The most widely used covalent binding strategy is the heterobifunctional crosslinking of the amino or carboxyl groups on antibodies to the free carboxyl or amino groups using EDC along with NHS or sulfo NHS. Therefore, there is a critical need to optimize the concentration of EDC as well as NHS for EDC-NHS crosslinking of antibodies, which can substantially improve the analytical performance of Electrochemical sensor.
Please suggest the optimum concentration of both with a valid reason.
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Dear Kumar
You can follow this article
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Hi,
I am trying to conjugate ethylenediamine onto carboxylic acid at my compound. To do that i used EDC/HOBt or EDC/NHS reagents. In my all reactions, i obtained more polar spot than my carboxylic acid containing compound on silica gel TLC, mobile phase: 60:40:10 (CHC3: MeOH:Water). When i quenched the reaction with water and tried to purify on reverse phase silica gel column chromotography by eluting methanol:water or acetonitrile:water systems, i did not get the ethylenediamine conjugated product.
Is it possible to convert ethylenediamine conjugated compound to carboxylic acid containing compound by adding water?
Do you have any suggestions/ comments about the separation of ethylendiamine conjugated compounds?
Thank you for your help.
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"Is it possible to convert ethylenediamine conjugated compound to carboxylic acid containing compound by adding water?"
Short answer, no.
When you activate your carboxilic acid with EDC, you form an O-acylisourea intermediate, which will be attacked by either i) an amine group from ethylediamine, forming your amide of interest; or ii) the OH from NHS, to form the NHS-ester, which will be subsequently be attacked by an amine group from ethylenediamine, forming then your amide of interest.
Adding water deactivate the O-acylisourea, converting it back to your original carboxylic acid. However, nothing will happen with the amide you formed.
To hydrolise an amide back to carboxylic acid, you need a base or acid catalyst, and a lot of heat, which I am assuming is not the case.
If you are having problems with your reaction, make sure that you have dry reagents and dry solvents, to not deactivate your intermediates.
"Do you have any suggestions/ comments about the separation of ethylenediamine conjugated compounds?"
That does depend on the carboxylic acid chain / side groups from your starting material, but increasing the pH of your system might increase the solubility of your carboxylic acid in water, aiding in the separation.
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Hello,
Below is the protocol which I have been using.
In brief, PLGA-COOH (10ug/uL) was dissolved in DMSO.
EDC (400mmol/L) and NHS (100mmol/L) dissolved in DMSO were added into PLGA solution and the mixture was stirred for 1 h.
Then, 10x excess methanol was added to the solution to extract the activated PLGA.
The activated PLGA was dissolved in DMSO.
Aqueous NH2 aptamer (100uM) was added to activated PLGA solution and stirred overnight at 4 C.
10x excess methanol was added to the solution and centrifuged to get precipitate.
But, the problem is the yield of precipitate formed is extremely low.
Is there are any errors on the protocol? Any advice is greatly appreciated.
Thank you.
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Appreciate your suggestions. Will contact the referred person for more informations.
I am actually looking for an effective method for conjugation reaction to be carried out in solvent instead of water, due to the water insoluble PLGA. TQ
Regards,
Hock Ing
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I'm searching for the reaction between an activated NHS ester and cyclic alcohol. The alcohol (cyclic one)should attack the ester carbon, and N-hydroxysuccinimide should be released.
I have tried to make an ester directly from the carboxylic acid and cyclic alcohol, in the reaction with EDC and DMPA in dried THF but unsuccessfully. Also, before that, I've tried to synthesize the same ester in two steps. In the first one, I synthesized an acid chloride which was supposed to react with cyclic alcohol, but the reaction was unsuccessful too.
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Often such reactions fail due to traces of water present. Not only the solvent needs to be dry, but all other reagents, too. Air moisture is detrimental, also.
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Hello everyone,
I need to couple a PEG24 spacer to a CRGDfk peptide, not on solid phase, I tried two times the reaction using EDC and NHS to activate the PEG carboxyl, using MES and PBS buffers and adjusting the pH, as reported on the literature, and cleaned through dialysis, but it didn't work both times.
Does anyone have any suggestions? Should I use DMF instead?
Thank you in advance
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Most dialysis membranes would not keep your product inside, because your product is too small. I assume that you mean Cyclo(-Arg-Gly-Asp-D-Phe-D-Lys)? It is much easier to purchase a preactivated PEG24-NHS ester and conjugate it at ca. pH 8 in phosphate buffer. A purification on reversed-phase HPLC might be preferable. The conjugation should be confirmed by MALDI or other MS. In some cases, you might use the product without any purification, since a small excess of PEG24 should make no effect in many experiments. If you prefer to activate the PEG24 carboxylic acid yourself, a conjugation in dry and pure (!) DMF, usually sold as "conjugation grade", might be performed.
You should use dicyclohexyl carbodiimide DCC (byproducts lead to a precipitate) or freshly (!) opened EDC. EDC degrades quite easily by traces of air humidity. NHS also needs to be as dry as possible or add DSC (disuccimidyl carbonate) as a chemical drying agent.
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This is a general query regarding the activation of the carboxylic acid group using EDC/NHS. As far as my limited information I came to an understanding that lowering the PH while activation is essential when proteins are to be activated and functionalized. But in the case of non-protein compounds which does not have -NH2 group and possess only -COOH group, will lowering of pH be any useful? as chances of having self-interaction/cross-linking will be near to none.
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Activate your carboxylate containing small molecule by using EDC/NHS in 0.025M MES/NaOH buffer, pH 4.7 for 30-60min (may need to find optimum incubation time) then add the activated molecule to your amine- containing protein/ molecule made up in 0.2M sodium bicarbonate buffer, pH 8.3. 4-24 hour incubation should work .
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We are trying to build an electrochemical sensor using the EDC/NHS linker on a screen-printed gold electrode to detect human serum albumin.
We are able to observe the impedance change from the bare electrode to EDC/NHS linker layer without a problem, but when we add antibody and antigen on the electrode the impedance decreases.
Based on what we read in the literature, adding antibody should increase the impedance, so I am very confused.
I would really appreciate any advice you could provide us regarding what we could try to solve this issue.
our general protocol is as follows:
1. Prepare electrode
2. incubate in MPA for 30 min
3. incubate in EDC/NHS for 30 min
4. put antibody on the working electrode and incubate in the fridge overnight
5. put HSA on the electrode and incubate 1 hr
between each step we rinse the electrode and measure impedance in Ferricyanide/ Ferrocyanide
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The sodium azide might interfere with the NHS chemistry. Try to exchange the buffer. The pH of 7.4 is a little bit low. A pH of 8 is often seen as optimal. In addition, be aware that the concentration of the antibody needs to be higher than you might expect. Most researchers use a MES buffer of about pH 5 for the EDC/NHS step.
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I have to conjugate an amino modified oligo with an NHS ester modified polymer, and I'd like to check the efficiency of my conjugation by detecting the unreacted groups.
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A typical test for free amines is the Kaiser test - it is described for resin-bound peptides, so you will need to check wether it can be used for molecules in solution. Sigma-Aldrich product and protocol: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Datasheet/1/60017dat.pdf