- Dominique Liger added an answer:10Is it possible to purify protein from SDS-PAGE in active state?
someone have any protocol related to purify protein from SDS-PAGE in native active state.
The recovery of an active protein from a SDS-PAGE is more than unlikely as SDS is used at effective denaturating concentration, samples are boiled in loading buffer in order to destroy any kind of structure but the primary one before loading onto gel.Following
- Marco Bauzá Thorbrügge asked a question:Newcould we get RNA from ethanol preserved samples? Could you recommend me a RNA/DNA isolation kit?
The samples are Prawns muscle and are stored in ethanol 100% for a day at RT and later stored at -20ºC (for example... 2 weeks))
I want to isolate RNA and DNA, just for a simple quantification and maybe later do RT-PCR.Following
- Adam B Shapiro added an answer:1How will I get to know that My DEAE seaphrose column is 'OK' with binding properties? which protein should I have to take to check this problem ?
I am doing purification of T7 polymerase with DEAE seaphrose column at buffer pH 7.9 but it is not binding with column .
There are several reasons why a particular protein may not bind to an anion-exchange column. (1) The pH is too low, causing the protein to have a net positive charge. (2) The ionic strength is too high, such that the salts compete with the protein for binding sites. (3) The binding sites on the column are saturated with something else, such as nucleic acid or other proteins, already. (4) The protein is aggregated.
I doubt that problem (1) is the cause, since the pH is already pretty high, although I don't know what the pI is of T7 RNA polymerase. Problem (2) can be solved by dialyzing the sample against a low-salt buffer and/or diluting it with a low-salt buffer. Problem (3) could arise from inadequate washing of a previously used column, or a column that is too small for the sample that is being applied to it. To check for problem (4), you can do gel filtration chromatography.Following
- Esra Balıkçı added an answer:5Is BL21 able to get expression of EGFR tyrsoine kinase domain?
I am working on purifying EGFR tyrosine kinase domain.
I have done cloning its tyrosine kinase domain (669-1200 Amino acid site) into pGEX-2TK vector.
I have problem that protein was not induced in 0.1-1mM IPTG for 30-37C.
BL21 is not good for purification of EGFR tyrosine kinase domain?
If you have experienced as my problem, please comment what I have to breakthrough for my problem.Following
- Sebastian Schmitt added an answer:5What if i still have Protein contamination in my DNA samples even after purification by using kit?
I did nanodrop to check the conc of stock DNA after purification by kit but it still shows a ratio of 2.1-2.2Following
- Adam B Shapiro added an answer:1Has anyone faced the difficulties in dissolving the pellet of total protein in buffer?
I am extracted total protein from Rice plant by Phenol extraction buffer followed by overnight precipitation in ammonium acetate, pellet down the pellet, 3 times washing with methanol, 2 times washing with acetone and then air-drying of pellet. I have to dissolve this pellet of protein in 20mM ammonium bicarbonate solution, but it is not getting dissolved. I also used 1X PBS and 20mM Tris buffer but the pellet is not dissolved properly.
Anyone dissolved protein in ammonium bicarbonate solution please suggest me regarding this, if any modifications needed. I have to move further with ammonium bicarbonate solution.
Since the proteins are completely denatured by phenol extraction, I think it is unlikely that they will dissolve in a simple, fairly neutral, aqueous buffer like ammonium bicarbonate. You might be able to dissolve them in SDS-PAGE sample buffer.Following
- Paul Rutland added an answer:2How does a FITC containing-PCR product run on an agarose gel?
I am running PCR products of different size on an agarose gel, and I get bands that indicates products shorter than expected size. ( for your info agarose gel 1.5% and PCR product of expected size of 500, 1000 and 3000bp, Yes I am using the right agarose concentration if you are doubting).
Since all of them are derive from using a forward primer that is FITC conjugated, I am having the doubt that FITC "slow down" the run. Is it correct? Does anyone have any experience with FITC (or any dye conjugated primer)? Thanks for the help.
FITC as suggested above would have to speed up the mobility of DNA to appear smaller. FITC does not change the mobility of proteins in PAGE gels so i think there may be another answer. If you are happy with your size standard then maybe there is a different salt concentration in size standard compared with pcr samples which might make a difference in the mobility in an agarose gel...are you using large amounts of betaine ( Q solution) or other salt in your PCR?. Can we see a gel picture please? If it were just the larger band being the wrong size I might suspect regions of homology causing the dna to loop and have secondary structure problems which running in denaturing PAGE would eliminateFollowing
- Faruk Abdullah Saurav added an answer:2How can you improve your 2D gel?
I am trying to run 2D gel. I am extracting protein from mammalian cell by using RIPA lysis buffer and then precipitate the protein with acetonitrile, then re suspend the protein in 250μl of re-hydration buffer sonicate the sample for 10mins and then start IEF focusing and so on by using invitrogen protocol and this is what I get every time. Can someone suggest me any solution on how to improve the gel or the whole procedure?
Thank you for your suggestion Dirksen but I have already tried that, the image I shared was after applying that suggestion. Thank you again.Following
- Grant Shimamoto added an answer:6Does Sucrose in Lysis Buffer flaw inclusion body purification?
I add sucrose to my lysis buffer (E. coli culture) in order to prevent aggregation of my target protein during lysis. When I centrifuge the lysis mixture (13000 g for 15 minutes), I get my protein in the supernatant (at least seemingly, checked by SDS-Page).
I now suspect that the sucrose solution, due to its high density (I use 1 M sucrose) hinders the inclusion bodies from settling down during centrifugation and that I therefore rather detect floating inclusion bodies, and not truly soluble protein.
Can anyone confirm this effect? What can I do to circumvent this, if it poses a problem? If not, I'm happy, since then I would really have soluble protein :)
Thanks for all answers,
What is the scale of your process and do you have access to dynamic light scattering (DLS)? If your scale is in hundreds of milliliters then ultracentrifugation may not be convenient otherwise Adam's point is applicable. Is the lysis method by homogenization or lysozyme/hypertonic solutions? After your first 13 K x g spin a DLS may help to assess particulate distribution as well as aggregate sizes in the supernatant. Is it possible to use macro, micro, or ultrafiltration methods in a TFF mode? Is it possible to substitute low concentration mixtures of arginine and glycerol for the sucrose to avoid the high density but yet stabilize the protein?Following
- Christopher S Morello added an answer:5Is there any way to store linear DNA product that I get from vector digestion as well as the PCR product so as I can keep them as a backup?
I am about to perform a protein expression experiment and right now what I have is my gene in a pUC57 vector. As I'm not experienced in the procedure, I wanted to ensure I had backup DNA to work with (it's expensive!). For this reason, after digestion I am going to perform a PCR experiment on the linear DNA. My question is: is there any way to store the linear DNA product that I get from vector digestion, as well as the PCR product that I do not ligate into another vector and transform so as I can keep it as a backup? Would -20degC do?
All of these are good suggestions, especially the one about having a midi "seed" stock of your plasmid that you use only for transformation and propagation of more of the vector. All of the storage ideas are good too, but I think -80 degrees C for storage is a bit overkill, but may have been helpful for certain vectors.
That said, on several occasions over the years, I knew I was going to be subcloning several (5 - 20) viral genes into the same 2 or 3 expression vectors for DNA immunization. These were all for blunt-end ligations to keep reading the reading frames for fusion proteins intact, so I didn't have ssDNA sticky overhangs to worry about. I linearized a large amount of vector at the multi-cloning site, phosphatase treated it like crazy, and then gel purified the linear fragment away from the small proportion of uncut plasmid that was usually contaminating denatured, supercoiled vector that was resistant to digestion.
Once those vectors were quantified, I kept them at 4 degrees C and they gave high ligation efficiency and low background (vector alone ligation colonies) for years. I did the same procedure with sticky ended vectors, but probably didn't keep them at 4 degrees for as many years so I probably wouldn't have noticed much decrease in efficiency. The concentration of these stored vectors was usually 200 - 400 ng per uL in TE8 or 10 mM Tris pH 8. So, I'm from the camp that I avoid freeze-thaw cycles wherever possible.Following
- Simonetta Ulisse added an answer:24A problem of "disappearing" protein during concentration of the sample: can anyone help?I am producing protein in baculovirus. My protein has a His-tag and I am purifying with Ni-NTA. After obtaining elution (with 200 mM of Imidazole) I took a bit of the sample and I made bradford to know what is the protein concentration and in which eppendorf is present the protein. I Froze the Elution. When I thawed the elution, there is a white precipitate that is not soluble even in 2, 4, 6 or 10 M UREA. Is this the protein? is it possible that it is not soluble even with a high concentration of UREA?
After concentrate the protein, I lost almost all sample and It is not present in the filtrate.
I think the protein is joining at the membrane (supposedly, low binding protein), but I tried to dislodge it with urea and I did not find it.
Can anyone help?
you can add L-Arginine in eluition buffer during purification in column to prevent aggregation and sarkosyl , after concentration and before freezing, to separate soluble and insoluble protein fractions.
- Sarang Mahajan added an answer:11Can someone suggest a way to elute biotinylated proteins from avidin agarose beads?
Generally in all the elution protocols, the proteins are eluted in very harsh conditions. E.g Boiling in SDS sample buffer, elution by using Guanidine HCl. This creates a lot of avidin background when run on a gel. I have to submit my sample for LC MS. In order to do that I need to reduce the noise. Can anyone suggest me any method that can reduce the background noise?
I haven't done it anytime. If it works with you please let us know.
- Mick M Welling added an answer:7Why is my protein precipitating after fluorescent labeling?
I'm trying to label a small 16kDa protein with an Alexa maleimide dye. However, after dye conjugation almost all the protein precipitates out of solution and/or becomes inactive. The protein is soluble and active before labeling. I've tested different buffer conditions (pH, detergent, salt conc, reducing agents) and labeled at both the C-terminus and at another cysteine residue I mutated in. I'm using an intein-CBD affinity tag to purify my protein. Has anyone run into this issue before?
Try a more hydrophilic dye with the same Ex/Em characteristics; you are precipitating your protein because of making it less water soluble. Success!Following
- Syed A Ali added an answer:1Impact kit ptyb21 to purify eucariotic protein?
Hello, Is somebody using impact kit ptyb21 to purify protein? I am triyng to optimize the protocol conditions, but I cant get the purified protein.
Thanks in advance.
We have tried it with some eukaryotic and viral proteins but did not work in our hands. After wasting (not really, for we always learn) so much time, I have finally come to a conclusuion that it is best to use eukaryotic systems for the expression of eukaryotic proteins. Optimizing conditions to express eukaryotic proteins in E. Coli sometimes take lot more time (and resources) than expressing those in eukaryotic systems.Following
- Tanmoyita Nayak added an answer:3What is the best buffer for maintaining transient nuclear protein interaction?
I am trying to find interacting protein partners for a small nuclear intracellular domain (which is my protein of interest). My plan is to do IP with nuclear extract followed by Mass spec.
Found several options in literatures but confused in choosing the proper detergent. Urea or SDS would be too harsh for maintaining weak interaction in my opinion.
Could anyone suggest me a good detergent for nuclear protein extraction that will lyse the nuclear membrane but at the same time would keep the weak interaction between proteins as well.
Thanks in advance.
Thank you for your answers . I will keep that in mind.Following
- Markus Gerhard added an answer:6How might I increase protein yield and solubility for helicobacter pylori recombinant proteins?
I am trying to express and purify a helicobacter protein. I had tried cloning and expression with CT his tag(pET21c+) but the protein was in hard inclusion bodies. So I changed my tag to GST (pGEX). Expression is fine but mostly protein is in the pellet. Can changing the bacterial growth medium from 2X YT to TB help? is there any other way to increase protein solubility and expression. I am using the Rosetta plysS strain for expression as it showed better expression than bl21plysS.
Before trying such changes, it would be important to know which kind of Protein you are trying to express. Does it have a Signal Peptide? Maybe a Membrane anchor? These must be often removed. One or two amino acids can make a Major difference. You may send me the ID or sequence, and we can have a look to propose you an optibal Expression construct.Following
- Lax Lee added an answer:4Pull Down protein interpretation?
I am doing pull down using His tagged protein A (28kDa) and an untagged protein B (47kDa). I incubate the proteins in presence and absence of a drug. In presence of drug protein A should interact to B. Otherwise there is little interaction of these proteins. What should be the result of this pull down? Should I see the pulled protein @ 47kDa using the specific antibody for B? That is, a better intense band compare to minus drug?
What I could see in the coomassie stained gel is, better band in + drug @ 47kDa area. Does that mean pull down worked? I also got something around ~75kDa (would this be the complex of protein A and B). I normally elute using 300mM imidazole and incubated @ 95 degree in SDS loading buffer. So the proteins might dissociate. I am waiting for the WB result soon. Please give your suggestions.
Thanks Venu. Currently my binding buffer contains 5mM Imidazole. and my wash and elution buffers contain Imidazole as well. I have proper controls too :) Thanks again.Following
- Richard Christison added an answer:1What kind of lysis buffer can be used for the insect cell?
I expressed one protein in insect cell using bac-to-bac system. The protein was secreted in 293T cell. But it was stucked in cytoplasm. The protein was aggregated when it was lysised with different lysis buffer even resuspension with high concentration salt, 2-ME and Urea. It only precipitated with 5000g for 20 min. It could not be loaded to the column. How can I solve this problem?
Hi Yi Wang,
I'm a bit confused as to what you have done here.
Have you tried to express a baculovirus in 293T cells? 293T cells are mammalian and you will not get expression in them from a standard baculovirus.
Or are you saying that a protein that expressed and secreted in 293T cells doesn't do so when expressed in insect cells? If so, which insect cells? High5s sometimes give secretion when Sf9s or Sf21s don't.
As for lysis buffer, this is really a protein question and could require screening of a range of buffers, pH and salt conditions. However if the protein is already insoluble in the cells the buffer conditions will not help make it soluble short of going down a refolding route.
More information as to what you have done will help.
- Mehtap Kara added an answer:3Shoudl I use tissue samples that I thawed once and froze them again for protein carbonyl analysis?
I want to analyse protein carbonyl assay on cardiac tissue, but I had to thaw the tissues once and freeze again. Is it possible to do protein carbonyl analysis?
thank you all very muchFollowing
- Nick Demarco added an answer:4Does anyone have experience with the Akta Start?It seems that it can do the same purifications as the higher end Akta Purifier/Pure/Avant, it's just that the pumps are peristaltic instead of piston based, but the price is much better!
The Start is a limited system because of its low pressure (0.5 MPa, 5 bar, 75 PSI) and low flow rate range (0.5 mL/min to 5.0 mL/min). Gradients are accurate within 10%. It can only run columns like 1 & 5 mL and . HiTrap columns and their many equivalents like BIo-Works BabyBio and Practichem's Reza columns.You will not be able to run many larger (longer) columns on the Start due to the pressure limitation. The Size exclusion / gel permeation may not be possible at all. Source: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1380023203850/litdoc29027051_20140901213218.pdf
Although alternatives like Practichem's Arista Slice are substantially more expensive to purchase, they run every technique and column in use today. It's smaller than the Start and runs on a browser. Disclaimer: I'm the president & founder of Practichem.Following
- Sven Manfred Lange added an answer:1Does anyone have a pET vector construct with 3C protease cloned in?One could buy the enzyme from company but we would like to purify it in lab itself. Ideally, I would like to have the protein tagged with GST but 6X his tag would be fine too. Hoping for a positive response.
FYI, the 3C protease cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) is also recognized by it's engineered variant better known as PreScission protease.Following
- Peter Rehbein added an answer:4Is there any method other than column chromatography for purification?
In case of solids we can use washings and precipitation methods to purify from its impurities after synthesis process other than column. I want to know if there is any other method or methods except column to purify or isolate a pure compound which is in liquid form after its synthesis if yes then how?
Ammonium-Sulfate fractionating precipitation.
There is a multitude of methods, for a decent overview I recommend you refer to a basic textbook on protein purification methods.Following
- Engin Yapici added an answer:5Can I use swinging bucket rotor to centrifuge bacterial cell lysate for protein purification?
I am going to purify a GST tagged protein from bacteria. I don't have a fixed angle rotor in the lab at the moment to centrifuge the samples after lysing the bacteria. I only have swinging bucket rotors and the max speed I can go is 4700 rpm (4816 g). Has anyone tried to use a swinging bucket to centrifuge bacterial cell lysates?
Thank you for your answer. Can you please elaborate it a little bit? Why do you not suggest it? What do you think might happen? Have you tried it before and didn't it work?Following
- Andre Fernandes added an answer:7What are some advise for isolating an enzyme from a cell line?
I am trying to isolate the enzyme 5a-reductase from my PC-3 (Prostate Cancer) cell line. Does anyone have any advice to give concerning this extraction?
For your kind advice, please.
The only two minor side effects that I can foresee are:
1. The cells could already produce a normal inhibitor that could compete or intensify the effect of your compounds (I don't know if they do but it's a possibility). You could try to avoid this by comparing how your compouds act on normal AND cancerous cells. You could also try to enrich your extract using only one chromatography step like ion exchange or size exclusion. If you do this "clearing" you could eliminate a possible natural inhibitor.
2. Your assay could detect the activity of other reductase. I don't know the specificity of your assay (Does it work only for 5alpha or the substrate can be processed by different enzymes?). In this case you could change your question and investigate total reductase inhibition OR maybe try to knockdown each one of them (I believe there are 3 isoenzymes but I'm not sure) using siRNA.Following
- John Schloendorn added an answer:3What should be the optimum protein load (volume and concentration) when purifying a protein by sephadex G 100 , Column chromatography?
What should be the optimum protein load (volume and concentration) when purifying a protein by sephadex G 100 , Column chromatography
Any suggestions would be highly appreciated :)
The protein amount (as mass or moles) is not really a key variable for these kinds of columns. A broad range will work, as long as your detector can see it.
Resolution will be better a smaller amount of protein (assuming constant volume as others have discussed). You should try a few different amounts and see for yourself. There's no good substitute for experimentation here. It's too dependent on your situation.Following
- Antonio Trani added an answer:3I am having trouble when purifying peptide on a semiprep column, the injections are not consistent. Any ideas?
I am purifying my peptides, easy linear 6 to 11 AA peptides, on a semi-prep column ( C18, 130A, 5um, 10 mmx250mm) but I am having trouble because my first injection is OK, but starting on the second one, the peptide elutes in the front. Any ideas? I have checked all the solvents and the HPLC, the pressure is stable, etc.
You need to equilibrate the column for at last 10 column volume. That means, if you use 25 ml/min you will need 6 min at the initial condition before the new injection.Following
- Konrad Miatkowski added an answer:7How do I couple proteins to cnbr-sepharose for making a protein specific resin?
Firstly, I wanna purify one kind of antibody from serum which is specific to one kind of protein. Ex) protein XX. So, I coupled this protein XX to CNBr sepharose resin to make protein XX affinity column. I coupled 1 mg of this protein to 0.75 g CNBr resin, but when I checked the protein XX column's affinity, I found the capacity of this affinity column is so weak to bind with anti-protein XX antibody in serum. Then I wanna know what's the problem? If I coupled so small amount of protein to CNBr resin?
We also would start with 5mg of protein per 1 ml of resin and most of the time we would get about 0.5 mg per 1.0 ml of resin capacity. Occasionally we would go as high as 10mg/ml and as low as 2-3 mg/ml of resin to address various binding issues.Following
- Harir Mohamed added an answer:2Tyrosine substrate
I performed Tyrosinase purification from bacteria broth medium , but I have difficulty to purify this enzymes because of the presence of melanin. Does someone have any idea on how to remove the melanin without affecting the enzyme activity?
Der Pr Adam,
Actually I didn 't try it ; I have just starting Dialysis in pahosphate buffer 50mM Ph 6.5