Questions related to Peptides
I've been trying to make a solution out of DMF, Fmoc-osu and Glycine. For protection of glycine. I have a hot plate with a magnet and an ultrasonic machine. But nothing is working! I have also been trying to slowly add water to the mixture but Fmoc-osu precipitates as soon as i mix it. And it turns into a big mess. Please help me!
I am currently conducting research that involves the use of PatchDock Unfortunately, I have been unable to obtain the necessary documentation and software from the PatchDock team.
If any of you have access to the PatchDock software or its documentation, or if you could provide guidance on where I might be able to obtain it, I would greatly appreciate your assistance. My goal is to establish the module in our HPC system for our ongoing research.
We need to accurately measure the protein/peptide concentration in a sample that is at least 80% DNA (primarily ssDNA less than 80 bases long with some dsDNA also). A majority of the methods I've looked at (Lowry, Bradford, NanoOrange, etc.) will not work with such a high DNA content. We also don't want to add any proteins (like DNAse) to get rid of the DNA because that will affect our measurement of protein contamination.
Any suggestions are welcome. Thanks!
I have a collection (around 500) of peptide 3D structures (PDB file each 10 residues long and for a given sequence). I need to cluster it based on RMSD values among them. Is there any Python module or any other software which could do that and identify the distinct structural clusters among those 500 files?
I'm researching platelet-derived growth factor signaling, and recently ran into a big problem. Specifically, I am determining whether a particular peptide can activate the PDGF receptor. My initial results using western blot and immunofluorescent microscopy were very promising; compared to negative controls, I saw significant receptor phosphorylation after treatment with recombinant PDGF-BB, a peptide previously shown to activate signaling, or my experimental peptide.
However, my results are suddenly no longer reproducible. I now see no change in receptor phosphorylation or downstream pathway activation between any of the treatment/control groups. I have been troubleshooting for months, and have tried different media, different cell types, fresh reagents, different harvesting methods, and different timepoints. For reference, most of my experiments have been conducted at a PDGF-BB concentration of 100 ng/mL, and peptide concentrations of 1 ug/mL. Cells utilized include THP-1 macrophages differentiated using PMA, and HeLa cells. Timepoints have ranged from 30 minutes post treatment to 24 hours post treatment.
Based on literature, even if my experimental peptide does not activate the PDGF receptor, the PDGF-BB treatment should be a reliable positive control. Has anyone experienced a similar issue in which cells no longer respond to positive controls? Does anyone have any suggestions for conditions to test?
I am working on peptide containing microsuspension (particle size: 20-30 micron) which has to be sterilised, since it is a peptide molecule, facing challenges with heat sterilisation method. Please recommend suitable method.
Hey all! I have a question about my proteomics data evaluated in Proteome Discoverer. I got three volcano plots of three biological groups in a ratio with control group. In one group I see a strange pattern, while other two look normally. Log2 ratio is somehow 100% -related to p-values with no exception (please see the graphs). Obviously it is not the issue of plotting itself, but in calculating the ratio or the p-value. Quantification was done using non-nested design, label-free quatification, pairwise-based ratio calculation, t-test, normalization of total peptide amount.
Does anyone knows the reason for that pattern?
Thanks a lot!
I have developed an peptide with machine learning that targets receptors vegfr1, vegfr2, and vegfr3. I plan to test its binding specificity on each receptor and measure the downstream effects in a cell line
Hi I am going to test some peptides as positive control for my ELISA but don't know what sort of concentration to start of with. Any suggestion will be very much appreciated.
We synthesized a peptide made up of 7 amino acids. After cleaving the peptide with TFA, we tried to precipitate the peptide from cold ether. However, it seems like our peptide is somehow soluble in ether. Are there any other organic solvents to precipitate peptides?
I am testing out an europium labelled peptide for the ligan binding assay and was hoping to find the Kd through the saturation curve.
Can people explain to me why my saturation curve is a straight line rather than what it should look like.
I plan to perform the Fluorescence polarization experiment. I have some FITC peptides ordered from the company with purity > 95%.
My peptide is 15-20 a.a long and very basic with pI ~12.
I read that most people dissolve it in DMSO.
Can I dissolve my FITC peptides in water?
Hi everyone. So I have performed MD simulations on desmond of my protein bound to a mutant peptide? My objective is to elaborate how the mutant peptide is inducing conformational changes in the protein. How to answer the above question by looking at the RMSD? How can I explain the given RMSD in detail?
I am working on peptide molecule for SC delivery for my thesis work. Which solvents (or co-solvent) shall I use other than PEG, PG, Glycerin to increase the solubility (drug loading) of peptide molecule. Will combination of solvent would be beneficial? Or which surfactant can I add to it?
I am planning to use high pH reversed-phase peptide fractionation kit from pierce (Catalog no.84868) for fractionating labelled peptides. I have used off-gel fractionator (Agilent) to fractionate the samples in the past and the results have not been great.
Any reviews for the pierce kit based on personal experience that could be of use to me ?
I am trying to synthesize the following peptide, on automated peptide synthesiser: dabcyl-RAGGYIFS--edans. The sequence cleaves at dabcyl-GGYIFS-edans. This is what I see from maldi-tof. the peak for the truncated sequence has higher intensity than the required one. I tried using HATU, DIPEA and also PyPOB but the yield did not improve. I use the resin which has edans attached to it already. the HPLC results in poor yields of the desired product. Can anyone help with this peptide synthesis using EDANS-resin?
I am looking for companies to order a couple of custom peptides. From my search Biomatik appears to best the best.. value/service. Do you have any experience with this company?
Hi Friends, could anybody help with this query please? While doing the Bradford Assay I mixed 10 microlitres of my peptide sample with 990 microlitres of Bradford Assay and got the absorbance. So while doing the final calculation I should take into account this dilution and multiply by 100 to get the correct quantity of my peptide sample.
I am measuring PET-FCS of a peptide and after measuring for 90 min. I am noticing that there is no PET in the FCS figure. However, if I run the experiment for only 5 min, there is some PET. Also, the diffusion time decreases over time during the measurement. I am assuming that some kind of fragmentation is happening due to the excitation laser.
I will be glad if anyone can explain this fact or any possible theories are also welcomed.
Hi, I have been trying to dissolve a very hydrophobic peptide (containing cysteine) with DMF, however, the solution looks very cloudy when I add DMF, and further dilutions with water doesn't help much. Does anyone have any tips on this issue? In addition, I'm use NanoDrop to measure concentration, but when I use DMF as blank, it shows "bad blank". By the way, I'm measuring the concentration at A280. Thanks!
Can anyone explain why a study's ELISpot assays might be designed wherein its cells are being plated at a concentration of 200K/well (the standard amount) and then 100K/well? The concentration of the peptide being tested does not change, just the cells. We are somewhat blinded to what the study sponors are looking for, so I don't have the full details, but I struggle to understand what the point of this might be. Would a lesser concentration of cells be informative of the efficacy of the peptides? I would think that playing with the test peptide concentration would be better than that.
I am trying to dimerize a synthetic peptide (22 amino acids) with a N-terminal cysteine, that was added for this purpose. I use the BM-(PEG)3 crosslinker from Thermo Fisher, which is based on maleimide-thiol chemistry. I reduce the sulfhydryl-bonds using TCEP, add the linker and stop the reaction with DTT. All according to the instructions provided by Thermo Fisher. I check the results with an SDS PAGE, but so far the protein bands stay on the same height before and after the reaction. I tried to get a positive control with insulin, lysozyme and murine SAA, but only the SAA shows a very faint band that could be a dimer.
Has anyone used this linker successfully or has any tips on how to get the reaction working?
I have an acidic peptide with 12aa (containing 5 hydrophobic amino acids), disulfide bridge, Methionine, and amidation at the C-terminal. I have tried dissolving using acetonitrile, NH4HCO3 0.1 M, and 2.5% ammonia solution separately. However, I can still see some tiny particles in the solution, which is a little turbid.
How can I dissolve the peptide easily?
How can I be sure about the non-dissolved particles? Are they peptides or can they be impurities?
I have two synthetic peptides (20-25 AA length) with terminal cysteines that I have been trying to conjugate to maleimide dyes. The maleimide-conjugation protocol has seemed easy enough, but I'm struggling to prove that my peptides have successfully tagged.
I had tried gel electrophoresis with Coomassie Blue, but later found out there was not sufficient peptide in my sample for the bound-tag to show. I then moved to HPLC but my lack of expertise and a lack of appropriate UV detector on the machine I used meant that was unsuccessful. Most recently I had submitted my tagged sample to an analytical service at my university for LCMS analysis, but the results have been inconclusive because there appears to be excessive fragmentation (and no obvious cysteine-tag fragments, or any fragments that differ by an m/z equal to the tag/tag+cysteine etc.).
I feel like this really shouldn't be that difficult, but it's absorbed months of my PhD project and I'm still no closer to proving the success of the maleimide tagging. Any advice?
I'm running CHARMM36 simulations of a peptide on GROMACS and I need its N-terminus to be capped with an acetyl group. I checked the “aminoacids.n.tdb” file, but there is no entry for acetylated N-terminus. How can I find/make a terminus?
I am looking for help from those of you who work in peptide synthesis. I am preparing a peptide containing the following amino acids: HO-Cys(SAcm)- Asp(OtBu)-Pro-Gly-Lys(Boc)-NH2, by HRMS (mobile phase H2O/ACN + 0.1% FA) I observe a signal M=690.3, which corresponds to the desired peptide that lost a tBu protecting group from Asp. However, there is another signal with M=585.3, do you have any ideas of what it can be?
I need to produce a custom antibody and I need the company to also synthesize the peptide. Does anybody have experience with a company that they liked?
I was recently trying to calculate the concentration of amyloid beta concentration from the UV-Vis result. I found that it can be calculated based on absorbance at ~280nm with an extinction coefficient of 1490 M-1 cm-1. However, I did not observe such a peak in my sample at 280nm. Instead, there is a peak at 230nm (which should be the absorbance of the peptide?) I am wondering if I still could do the calculation based on this figure.
Any reply would be appreciated. Many thanks.
May I know how to calculate the persistent length of a peptide using MD simulation trajectory ?
Thank you in advance !
My peptide is Cholecystokinin (CCK8), MW=1142.35 (COOH-D-Y-M-G-W-M-D-F-NH2).
Stock solution in NH4OH 0.05M and working solution in acetonitrile.
I do MS infusion at conc. 500 ng/ml in acetonitrile.
I use two LC/MS machines: Micromass - Quattro Premier XE of Waters (Tamdem Quadrupole) and Applied Biosystems - API 3200 LC/MS/MS (triple quadrupole)
I run ES + but I can not see the peak at 1+, 2+, 3+,4+,...for [M+H], [M+Na], [M+K]
I wonder whether I have missed some other adduct ions that could be created during the ionization?
Or maybe my peptide is being degraded during preparing the sample?
Please give me some advice! Thank you!
I have been reading the following article on peptide structure prediction. https://www.biorxiv.org/content/10.1101/2022.02.17.480937v1.abstract I wanted to know whether alpha fold 2 can be used to generate distributions of structure of a given peptide sequence. In other words, Can one generate distributions of backbone Ramachandran angles of a given peptide sequence using structures predicted from various conformations generated by alpha fold2?
I have read protocols for development of antibodies from rabbits post immunization with peptide. However, I shall be the first to do this in our lab. Can anyone suggest how to perform (video/tutorial) the rabbit immunization step by step with precautions?
Hi everyone, I'm carrying out a peptide coupling reaction, with the coupling reagents being N,N'-Diisopropylcarbodiimide (DIC) & hydroxybenzotriazole (HOBt). I'm making glycine to couple with 3-chloro-2-methylaniline in the peptide coupling reaction. I have tried dimethylformamide (DMF), ethyl acetate, dichloromethane and acetronitrile, but glycine wouldn't dissolve in them. Kindly advise suitable solvents for the peptide coupling reaction or any chemicals that I should add to help dissolve the glycine. Thank you!
I am wondering if there is an easy data format to share the composition of a biochemical solution, for example a buffer consisting of certain concentrations of different substances, as well as some biologically active molecules (peptides, oligonucleotides, proteins, etc). Preferably the latter would have links to their accession numbers in different databases, or if that is not available, a FASTA sequence. I am seeking to improve the re-usability of data within the single molecule field.
How can I synthesize a peptide with N-terminal protection by the benzyloxycarbonyl group (Z-group) using Fmoc/tBu Solid-Phase Peptide Synthesis?
Can Z-Leu-OH be coupled during SPPS as the last N-ter amino acid? The peptide is 5-mer with the sequence: Cbz-Leu-Asp-Lys-Ala-Leu-OH.
Can we protect the N-terminal-CBZ group during peptide cleavage?
I´m unsure if the N-terminal-CBZ group is stable to 95% TFA treatment during peptide cleavage (& side-chain PGs removal) from the Wang resin.
Any reference would be helpful.
Thank you very much for your kind input.
I like to do SPPS by Fmoc/tBu chemistry to make a peptide (Z-Leu-Lys-Glu-Ala-OH) with an N-terminal-CBZ/Z group at Leu. I like to use Z- Leu-OH for the last coupling, but after the synthesis, I want to keep the Cbz/Z protection group at the N-terminus.
How to preserve the benzyloxycarbonyl (Cbz/Z) group at the N-terminus of the peptide during the removal of side-chain protecting groups with anhydrous TFA?
Covid-19 vaccination can induce multiple sclerosis via cross-reactive CD4+ T cells recognizing SARS-CoV-2 spike protein and myelin peptides
Qiu, Y.; Batruch, M.; Naghavian, R.; Jelcic, I.; Vlad, B; Hilty, M.; Ineichen, B.; Wang, J.; Sospedra, M.; Martin, R.. Multiple Sclerosis Journal; 28(3 Supplement):776, 2022. Article in English | EMBASE | ID: covidwho-2138820
I want to use a RALA peptide vector, and I have found that some studies centrifuge the plasmid/RALA complexes and resuspend the pellet before use, while others seem not to do so. It seems like the studies that don't centrifuge end up with smaller particle sizes than the studies that do centrifuge, but I haven't been able to find any definitive confirmation of this trend. Intuitively, it seems like spinning them at 10k RPM would mash them together to some extent, possibly causing aggregation/melding of the particles and lead to larger particle sizes after resuspension. The only advantage to centrifuging and resuspending I can see is that it would eliminate any toxic effects of free floating/non-encapsulated plasmid, but this wouldn't even really be a concern in vitro, right? Does anybody know of a study that has investigated this? Thanks.
I recently started to use peptides for cellular studies. Unfortunately, when I was designing the peptides I did not have a FITC tag to them (the peptide was synthesized by an external company). The peptides still have an -NH2 terminal that I can react with FITC-NHS pretty easily. But due to the small size I would not be able to isolate the peptide from unreacted/hydrolyzed FITC-NHS by MW cutoff as I would for proteins.
The peptides are not very cheap so I would seek alternatives than purchasing more tagged peptides. While I know I could purify it by HPLC followed by concentration / lyophilization, it's adding a lot of procedures to the pipeline and I'm considering if there could be alternatives. And so, I'm wondering if peptides could be precipitated by TCA/acetone precipitation similar to proteins?
While I believe the precipitation is dependent on the peptide structure, I used one of our peptides and tried it out - the precipitation worked (I saw visual white fluffs) and the peptide absorbance curve showed peaks at 214/280. The curve looked identical to the peptide solution (unprecipitated) but with a lower absorbance unit (22 AU compared to 5 AU post-precipitation).
My question is, is this way of isolating peptides legit? And if I used FITC-NHS, would FITC also be precipitated by any chance? Will the peptide lose stability / increase cytotoxicity during TCA exposure? I am not using the peptide for functional studies, just trying to visualize cellular internalization.
- If this way makes no sense I will refer to other methods. Thanks for any suggestions.
I need an advise, I’m using C7C PhD peptide library from NEB and after three rounds of selection and ELISA assay I sent my selected clones to Sanger sequencing. When I obtained the results I found out that the majority of sequenced samples (clones) had the sequence of M13KE vector without the insert of peptide library.
I wanted to know whether it is a common problem? Are there any steps to overcome this situation? Does it will affect the results of my selection? Why I got so many sequence of M13KE vector?
Sorry for so many questions, but I didn’t find anything about this in the literature.
Thank you in advance for advise.
I am thinking of inserting a small marker peptide into HDR fragments to detect and visualize CRISPR recombinant colonies.
Is this possible?
please guide me.
I wanted to have a rough estimate on time it takes to get the complete thermal distributions of backbone Ramchandran angles of a given peptide size of 18. Is it possible to estimate the time before doing the Monte carlo simulations. In the attached paper they have mentioned it took 108 CPU hours with 30 cores for 200 milliion CPU steps for TRP cage 20 residue protein. With this info is it possible to estimate the wall time of the MC simulations of a given biomolecule ?
We are developing a peptide ELISA for IgM and IgG detection.
We infected mice with parasite 1; for cross-reactivity analyses, we infected mice with two parasites (groups 2 and 3) and included a negative control group. Peptides were synthesized by solid phase peptide synthesis (Fmoc), with ~80% purity, from a native antigenic protein from parasite 1. Sera from different periods of infection were collected and frozen.
We coated the plates with BSA (40μg/mL) for 1 hour, and treated with glutaraldehyde 5% for 1 hour. Then, we added the peptide and incubated it overnight. Finally, we added glycine for 30min. We blocked the plate with PBS-BSA 5% for 1 hour, added the diluted samples and incubated for 1 hour under mild agitation. We added anti-biotinylated IgM or anti-IgG-HRP for 1 hour. For IgM, we added avidin-HRP for 30 minutes. For both, we added TMB for 10min, stopped with sulfuric acid and read at 450nm.
The results are not consistent inter-assays (low reproducibility). The ODs from the negative control group are higher than blank wells and similar to those from parasite 1. Sera from groups 2 and 3 resulted in higher ODs than those of the parasite 1 group. We did not expect this cross-reactivity, given the peptides appeared specific to parasite 1 in certain periods of infection and showed no homology to other parasites or microorganisms on Protein BLAST.
Does anyone have any experience with peptide ELISA that could help us?
We washed the plates after every step, as shown below:
•BSA: washed once with carbonate-bicarbonate buffer (pH 9,6)
•Glutaraldehyde: washed twice with carbonate-bicarbonate buffer (pH 9,6)
•Peptides: washed twice with carbonate-bicarbonate buffer (pH 9,6)
Glycine: washed once with PBS
Blocking and sera: washed 3 times with PBS-Tween 20 0,05%
Anti-biotinylated IgM: washed 5 times with PBS-Tween 20 0,05%
Avidin-HRP: washed 7 times, 5 minutes each, with PBS-Tween 20 0,05%
Blocking: washed 3 times with PBS-Tween 20 0,05%
Sera: washed 3 times with PBS-Tween 20 0,05%
Secondary antibody: washed 3 times, 5 min each, with PBS-Tween 20 0,05%
I was using fragbuilder module in python to generate peptides of sizes 4, 6, and 10. However, the issue with fragbuilder module is that some of the bond angles are deviating from the standard values. For instance, C_alpha--C--N bond angle standard value is 121 degrees but fragbuilder assigns 111 degrees. This angle deviation causes a deviation in the distance between the nearest neighbor C_alpha---C_alpha and its value is 3.721 angstrom and the typical standard value is 3.8 A. Also another bond angle is a deviation from the standard value by 6 degrees which is the C_alpha---C---N whose value is 111.4 degrees and typical standard values are 117 degrees. My doubt is how much deviation is allowed for MD simulations of peptides (or proteins) while fixing the bond lengths and bonds angles ?
Hello. For energy minimization of my peptide ligand, I am using Chimera software. Is it necessary to add hydrogen to the ligand prior to docking?
Example : I have the following sequence, of the penetratin peptide, conjugate with Cf:
The synthesis is done m=150mg resin, the resin substitution 0.65mmol/g.
I have got as crude after the synthesis m=134 mg. and I purify 20mg, I have got 4.88 mg.
Hello, I want to figure out the degraded product of a peptide (m.w.: >3500)). I did an LC-MS analysis and got a few degraded products (new mass). How can we get the formula or sequence of that degraded peptide from its mass (m/z)? Please share any online tools or materials that will be helpful.
Hello all, first post on ResearchGate!
I have been performing immunoprecipitations with FLAG beads (abcam, ab270704), eluting using SDS loading buffer (with DTT) and boiling. The eluted products are then used for SDS-PAGE and downstream Western Blots. We're concerned our proteins of interest are not being fully eluted from the beads using our current method. Abcam suggests a competitive elution using the DYKDDDDK peptide, however I'm having trouble finding it for purchase from them. Has anyone used that peptide product from another company and had success?
1 - In my last proteomics analysis I obtained peptides with PEP 0 ( zero). should I exclude them because it could not be calculated or it is almost zero and it is sure about the peptide sequences?
2 - I am using DDA to analyze my complex proteome. I am interested in a single protein. I run MQ using a single fasta file with my protein sequence. I obtain peptides with certain PEP. I am re-doing the same analysis but together with the fasta file of the single protein of interest I am adding the whole proteome of the specific organism. I obtain same peptides, with same intensities BUT with different PEP. Why? does anyone have an idea. PEP is not supposed to change and when I run with the whole proteome is actually lower then with a single fasta sequence.
Does anybody have similar issues?
I possess 250 peptides and one target protein, prompting my search for tools/servers that allow docking of multiple peptides simultaneously. While I have already utilized HPPdock, I am interested in exploring alternative options for this purpose. Are there any other tools available?
I am currently trying to characterize a small peptide that was designed computationally and later synthesized and purchased from a manufacturer. I want to perform Gas Chromatography-Mass Spectrometry in order to confirm its sequence first but unable to find any proper protocol of recent time has delayed my work. Can anyone help me with a protocol? Could be your own, used for a work and published. Thank you in advance.
I am trying to separate a small peptide (4 amino acids) synthesized using solid-phase synthesis after cleavage. I have tried dissolving the peptide in diethyl ether, but it did not percipitate from the cleavage reagent. What other methods can I use to effectively percipitate the peptide from the cleavage reagent and resin? Any suggestions or protocols would be greatly appreciated.
My experiment is to look at the intracellular location of a small peptide. I have difficulties when I try to do the IF staining for the small peptide that has been uptaken by cells, and the main problem is the loss of signal after permeabilization.
The peptide is around 6k Da and labeled with Alexa flour 647. It was added to the cell culture media for 10min, then, cells were washed and fixed with 4% PFA, then permeabilized.
I have compared the signals of the live cell, 4% PFA fix-only cells, and fix+permeabilized cells, they all were treated with same peptide the signal loss happened mainly after the permeabilization process, and especially at the cell peripheral part. In the fix and permeabilized cells, the signal of the peptide is only shown in the central part of the cells.
My first thought is, maybe due to low MW, the fixation is not strong or long enough for the peptide, so I have tried different PFA concentration(1%-4%) and time (5min-2h), but increasing PFA concentration and time did not help.
I also tried 0.1%, 0.5%, 1% TX100 and saponin for the permeabilization, it seems saponin is better than TX100, but still have the issue of loss of peptide signal.
I am just wondering if anyone knows the optimized or specific fixation and permeabilization protocol for this kind of application with small peptides. Thanks
after peptide synthesis then need use enough tfa cooktail to cleave the peptide from the resin. and how to easy separate the peptide and tfa is really bored work. especially when use much tfa.
can someone introduce any adsorbent easily extract the peptide from tfa cooktail ?
We are facing problem while doing cleavage. As we get final crude after using many combination of TIS Water & TFA and other was TFA TIS Phenol & DTT still not getting good results as impurity besides main peak was showing 7-13% and increases with increase in scale (determined by analytical hplc). Mass determination indicating it as +56 or 57 impurity determined tbu either not getting cleaved properly or getting reattached somewhere on amino acid sequence. But impurity fraction (the varying peak just beside main peak). The assumption of getting reattachment of tbu is correct or its some other impurity? And if yes then which it is.
I am trying to solubilize a peptide:oligonucleotide conjugate for intra-peritoneal injection into a mouse. At low concentrations, the mixture dissolves nicely in either H2O or saline, however at higher concentrations, everything crashes out. The peptide is hydrophobic but both the peptide and oligonucleotide are currently dissolved at very high (stock) concentrations in HBSS or TE.
I just tried Ethanol (10 and 50%), DMSO (10 and 50%), PEG-400 (10 and 50%) and Glycerol (50%). The saline and glycerol generally keep most of the protein:olgo conjugate in solution, but the others generate large solid pellets in the bottom of the tube after 30 minutes at room temperature.
Can anyone advise on other possible solvents to try?
I need help now. I run HPLC with one of the peptide, on RP with 0.1% TFA/ACN as mobi phases. There is an impurity peak came out. The weird thing about this peak is 1) did not show in my blank and system suitability standard. 2) peak area/height is not changing when sample is diluted. 3)did not show up in the dilution buffer inject (10x more), 4) this peak give the same area if I double the injection, though my peptide peak is doubled.
what could be the cause of this peak? Anybody have experience of this lipid problem in HPLC? I only have VWD detection.
Can a boiled peptide serve as a negative control in biological assays? or does it necessarily have to be a scrambled peptide of the same length?
I am expressing a fusion protein with a 6 HIS tagged eGFP SUMO followed by a small peptide. Because of the small size (~ 560 Da) of the peptide after cleavage from fusion, it is hard to visualize on any gel. I doubt that there is any cleavage happening at all. I wanted to know if expressing the fusion tag without any linkers between two proteins (eGFP and SUMO) makes it unrecognizable to the SUMO protease to cleave?
I am conducting a molecular docking study using peptides as ligands. I successfully ran the docking process using autodock vina, and when I viewed it using Discovery Studio, I realized that the ligand have missing bonds. I retracked my process and found out that the pdbqt file of my ligand is the problem. Here is a screenshot of my pdbqt file of my peptide, IF in DS. Attached also is the pdbqt file of my peptide.
Please help me on what to do.
I am developing a UHPLC method for content and purity for a peptide based on 100mM KPF6 and 100mM NH4PF6 pH 3.5. TFA as an ion pair reagent does not give the required separation performance. My question is less about method development HPLC for peptides, but more about the negative effects of KPF6/NH4PF6 in the UHPLC systems used (Waters IClass binary system and Thermo Vanquish Horizon binary system).
The negative effects on 3 instruments were already noticeable after 1 to 2 sequences (extreme pressure fluctuations):
The repairs carried out showed more or less the same damage:
- strongly porous seals in the pump heads
- damage to the pistons
- defective inlet/outlet check valves
One system was used over 2-3 months and showed the most damage.
I will ask my questions right at the beginning:
- What experience do you have in using KPF6, NH4PF6 as additive/buffer in mobile phases for UHPLC analysis or for method development for peptides?
- How can these reagents be used "safely" and robustly without causing damage to the pumps?
These chaotropic reagents seem to be highly corrosive. I would be glad to get some useful advice. Many thanks in advance for your support and advice.
Mobile phases are:
Mobile phase A 0.1M KPF6 pH 3.5 / ACN 8:2 (V/V)
Mobile phase B 0.1M KPF6 pH3.5 / ACN 35:65 (V/V)
Mobile phase A 0.1M NH4PF6 pH 3.5 / ACN 8:2 (V/V)
Mobile Phase B 0.1M NH4PF6 pH 3.5 / ACN 35:65 (V/V)
0.3M Buffer KPF6 pH 3.5
- 55.2 g KPF6, were weighed into a 2000 mL beaker. 1000 mL water was added and stirred until complete dissolution for 15 min. Sonification for approx. 2 min. It was adjusted to pH 3.5 with Orthophosphoric acid 85% (+/- 255 µL) and stirred well. The buffer was filtered with a Steritop-GP 1000 mL Express Plus PES 0.22 µm into a 1000 mL bottle.
Mobile phase A1: 0.1M KPF6 pH 3.5 / ACN 8:2 (V/V)
- 330 mL 0.3M_ KPF6 buffer pH 3.5, 470 mL water and 200 mL acetonitrile was transferred into a 1000 mL bottle and mixed well. Sonnification for approx.5. min.
Mobile phase B1: 0.1M KPF6 pH 3.5 / ACN 35:65 (V/V)
- 330 mL 0.3M_ KPF6 buffer pH 3.5, 20 mL water and 650 mL acetonitrile was transferred into a 1000 mL bottle and mixed well. Sonnification for approx.5. min.
0.3M Buffer NH4PF6 pH 3.5
- 48.9 g NH4PF6, were weighed into a 2000 mL beaker. 1000 mL water were added and stirred until complete dissolution for 5 min. It was adjusted to pH 3.5 with 25 % NH4OH (+/- 340 µL) and stirred well. The buffer was filtered with a Steritop-GP 1000 mL Express Plus PES 0.22 µm into a 1000 mL bottle.
Mobile phase A1: 0.1M NH4PF6 pH 3.5 / ACN 8:2 (V/V)
- 330 mL 0.3M_ NH4PF6 buffer pH 3.5, 470 mL water and 200 mL acetonitrile was transferred into a 1000 mL bottle and mixed well. Sonnification for approx.5. min.
Mobile phase B1: 0.1M NH4PF6 pH 3.5 / ACN 35:65 (V/V)
- 330 mL 0.3M_ NH4PF6 buffer pH 3.5, 20 mL water and 650 mL acetonitrile was transferred into a 1000 mL bottle and mixed well. Sonnification for approx.5. min.
After sequence, the systems were flushed with
- 85min with ACN/H2O 15/85; 0.4 mL/min
- 15 min with ACN/H2O 75/25; 0.4 mL/min
- 15 min with ACN/H2O 50/50; 0.4 mL/min
- following a low flow ACN/H2O 50/50; 0.1 mL/min
If we have Peptide sequence, can we develop correct DNA sequence from that? If Yes, what can be most suitable process? What can be the pros and cons?
Your answer will highly be appreciated.
I am testing different combinations of peptides grafted to alginate hydrogels. Inside these hydrogels I have culture human mesenchymal stem cells and now I want to see how the different combinations of peptide influence the cell behavior. For that, I would like to understand whether cell volume changes as well as cell major length, sphericity or other measurement that can describe cell geometry. I've been trying to use Amira-avizo to measure diameter/length (approximated from a 2D disc) of cells that have been 3D reconstructed. I already managed to get all these values but they do not correlate very well with the 3D reconstructions that I've obtained from my different conditions. Is there anyone ho know how to use this Amira for this purpose for this purpose or have suggestions for other software ?
we have synthesized 27Mer peptide which contains 8 glycine amino acid. and due to inefficient coupling one of the glycine coupling was partially completed giving us the peak of desglycine peptide.
The retention time in HPLC for the both is same. we have tried different methods , buffers and organic modifiers.
if anyone has encountered a same problem and solved it please let me know.