Hi, I am trying to express a GST-tagged protein using E.coli BL21(DE3) competent cells. I think this protein seems to be degraded even in the cells, because even I doubled the proteases inhibitor tablet in the sonication buffer and run the SDS-Page and Western for the lysate and the degradation still occurred.
I purified the protein using GST spin column following the manufacturer's instruction in 4C all the time and the result still showed very strong degradation(see the attached picture). Fortunately, I can see the target band which is at the top.
I have tried to optimize the expression that induces in OD600=0.5, 0.6 or 0.7 with 0.1 mM or 0.3 mM IPTG, then incubates in 16C or 25C for 2h, 4h or overnight, but none of the combination could reduce the degradation to a decent level.
Could you help me out? Thank you very much!
The photo appears to be of an x-ray film of a chemiluminescence Western blot. If so, what looks like degradation of your protein could be non-specific antibody binding. Have you stained the SDS-PAGE gel with Coomassie Blue to see the proteins directly?
Hi, Adam, Thanks for your input. Some previous gels yes, but this particular gel was not. Maybe non-specific antibody binding could be one of the reasons. I used the commercial Anti-GST tag primary antibody (1:1,000 TBST) and regular HRT 2nd antibody (1:30,000TBST). I used the manufacturer's recommended dilution factor. Probably, I could use 1:3,000 for primary or 1:50,000 for 2nd. What do you think? Thanks.
Are you using a Western blot, rather than Coomassie staining the gel, because you are sure the protein is degraded and you want to confirm that by showing that there are many GST bands at lower molecular weight?
Hi! Is the protein tagged at the C- or N-terminal end? If it is at the N-terminal, at least some of your extra bands could come from incomplete polypeptide synthesis.
Hi! Do you have a negative control (empty Ecoli lysate elution or elution of Ecoli transformed with phisiologicaly-neutral proteine cDNA) on the gel/WB?
Hi! What kind of lysis buffer are you using for your purification? I would suggest first to check all your buffers from beginning to end, and run all the lysis + purification at 4ºC. Also, how do you store your purified protein? % glycerol, temperature...
Hope it helps!
Hi!! Having a negative control will help you to see whether you have non specific proteins been recognized by your antibody during the western blot. Your buffers are very important and also the method you use to break your cells. Harsh sonication can damage your protein. We usually have glycerol and low or non detergent as it can affect our protein activity. Do you know your protein half life or stability? I have used OD= 0.3 and induced with 1 mM IPTG for 3 hours. This is faster but need to consider whether your protein aggregate or not. If nobody have purified this protein before consider that the Tag location can also affect protein activity. You might also try with the Tag at the N- terminal just in case.
My best advice is to use different buffers and perform mini scale purifications (pull down) to optimize your conditions.
Hope it helps!!!
Hi, Rosario and Janice,
Thanks for your answers. My lysis buffer contains 10% glycerol, 1% Triton X-100, 200ug/mL Lysozyme, 50mM Tris, 150mM NaCl, (pH 8.0) in 10 mL solution with two tablets of protease inhibitors. I used this buffer to resuspend the cells for sonication, 10s sonication treatment for 7 times with 30s intervals (until the cell suspend became half clear), and all procedures were done in ice (and there is no bubble produced during the sonication). I thought this condition was not that harsh, but probably do lower voltage (I used default setting working good for my other proteins) or less times of sonication treatment.
Do you have any specific suggestion regarding how to optimize my sonication/lysis buffer. One of my idea might be add some Zn2+ in it, because my protein is a Zn-finger protein, which may be stabilized by Zn2+.
Also, do you want me to boil it before running the SDS-PAGE? I used to boil the samples in loading buffer for one min, and I think this might be one of the reasons why I saw the degradation.
Thanks in advance.
As fas as I am concerned, GST always has problems of degradation. Themo Fisher says: " For reasons that have not been fully characterized in the literature, the structure of the GST-fusion tag often degrades upon denaturation and reduction for protein gel electrophoresis (e.g., SDS-PAGE). As a result, electrophoresed samples of GST-fusion proteins often appear as a ladder of lower MW bands below the full-sized fusion protein" therefore, maybe it is not a problem of the protein itself, but it is a problem of the analytical technique
Hi, I face the same problem nowadays, we tried different induction condition but still there are many band upon purification of GST tagged protein. Could tell us how you solved the problems? Thank you very much!
Hi, to get rid of your degradation bands is to simply have 2 tags. For example, N - GSTtag-protein-Histag - C you will easily get rid of all the degradation bands during your second affinity purification step. It is advised to first do the GST affinity purification and then His because His affinity tends to not be very clean but in principle it doesnt really matter. Incorporate precission (3C protease) cleavage sites at both tags to effectively remove both tags in one cleavage step.
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