I am using hek 293 mammalian system for flag and strept tagged secreted IgT production, I used direct blotting method and I got a smear in SDS-PAGE with a very faint expected size of my protein in WB, the smear was along the way of the protein migration, I suspected a proteolytic effect especially when cell viability was very low at protein harvesting day, approximately 55-59 %, do you have any recommendations to this problem. Thanks in advance
I guess you can use protease inhibitors for your application, too ...either commercially available protease inhibitor cocktails (Pierce, Sigma, Roth), or the single compounds such as AEBSF
Bestatin
E-64
Pepstatin A
Phosphoramidon
Leupeptin
Aprotinin
Was the smear above or below the expected IgT band
Above = precipitation of your protein
Below = proteolysis
You should consider growing a bigger batch of cells and harvesting earlier .... as there is no know way to reduce in-situ proteolysis of proteins (inside the cell)
what is r-protein? is it for receptor? if so maybe the smear you get is what you are supposed to see, as some receptor has modification of glucose, which when receptor is visualized it will appear as a smear.
Coctail protease inhibitor from Sigma should serve the purpose. Proteolysis inside the cell will be very difficult to stop. Only procedure I could suggest that one could fix the cells while harvesting.
Christoph Metzner, Much obliged, but do you think working with protease inhibitors will not impact my cell growth and protein production.
Thanks Glenn Soltes, actually it is above my expected band not below, do you recommend a solution to this smear, and what if this smear was due to the glycosylation heterogeneity since we produce Ig.
Amani Odeh, Hi Amani it is for recombinant protein (IgT), but back to the point of glucose if it was like that how could you solve this problem I heared using Tricine -PAGE may be helpful.
Thanks Utpal, If so, what about cell growth and protein production efficiency.
since it is recombinant protein , then it has no modifications, unless it was done via non-bacterial host. back to glucose modifications, i was expected back then that my protein had smear appearance, it wasn't a problem for me, i quantified it as it is. but this will not help you in this case sorry.. good luck
Hi Haitham
IgT and other Ig are generally very soluble and well behaved.
Yes some band heterogencity is associated with glycosylation, but at the molecular weight of Ig that usually doesn't smear up very far.
The most likely scenario is that you have insoluble material in your sample which is blocking up the well and causing smear up.
Fortunately the solution is very simple.
1) Filter your sample though a low protein binding syringe filter (0.45µ 13mm Millipore Millex PVDF Durapore HV filter SLHV013NL)
2) Use a fine gauge needle and syringe to draw the sample up and down 10x to break up any polymers like genomic DNA (if you think this may be present.
Also when you make a request for assistance of this type you should post a picture of your blot. It really helps.
Since the smear is higher than the expected molecular weight, I would tend toward the possibility of some precipitation having occurred in the sample. As the electrophoresis proceeds, more protein gets dissolved and enters the gel at different times. Streaking could also be due to too high of a load in those lanes.
Thanks Glenn Soltes for answer and recommendation, you are right I edited my question anyway, but actually we didn't use the ordinary electrophoresis loading our samples into wells instead we load our samples on a filter paper to soak the sample and electrophoresis is done on the surface of the gell. but anyway I will try to filter my samples. Thanks for kind help.
Thanks Dear Anna and how do you believe a solution could be ? Is it a filtration only or what ?
Haitham, I am familiar with sample application on isoelectric focusing gels but not with application of samples by filter paper on SDS-PAGE, so I can't suggest ways to help regarding the electrophoresis procedure. But I can suggest a few other troubleshooting steps.
1. Assuming you have enough solution, can you measure absorbance in a spectrophotometer at 280 nm before and after ultrafiltration? That will tell you if you have precipitation. If not, jump to #5.
2. If there is no precipitation, then you might get better results by loading less or loading using wells with a stacking gel. The stacking gel allows proteins to pass through easily so when they reach the running gel with the smaller pores, the proteins stack up in a tight band.
3. If you do have precipitation, the clarified solution might work.
4. However, if you find that most of your protein of interest is in the precipitate, you might want to look at different methods of getting the protein into solution, even possibly refolding. I found a useful practical review on this subject.
file:///C:/Cache%20Photos/Cache%20Family%20Photos/Kath%20and%20Chris/Kath%20and%20Chris%20in%20SF/biomolecules-04-00235.pdf
There are commercially available kits for the more common methods for protein refolding that are suggested in this article.
5. If you can't do #1, then try a lower load. If that does not work, then assume it is precipitated and try refolding the protein (#4).
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