I am doing purification of enzyme from culturables.Problem is that after GFC when i am running the protein on gel,band is not visible but the protein is there as when i am performing assay and zymography, activity of enzyme is observed.Specific avtivity is increasing by 100 folds but conc. of protein is so less that i cannot see the band on gel.I also performed silver staining still no positive result. Please suggest.
Silver staining is the most sensitive way to detect proteins on a gel. Coomassie stains are less sensitive. The only method that could beat silver stain is to carry out in vivo labelling with 35S methionine before you extract, then purify and do autoradiograms of labelled proteins, but nobody would consider this as it is too much hassle. If your zymograph is positive, you know the molecular weight of your protein on the native gel, and you have an assay to follow the protein, so your only option is to scale up the starting material so you have more of your purified protein after the various purification steps. Gel-filtration leads to dilution of the sample, it is always advisable to do a concentrating separation (ion-exchange or hydrophobic interaction with gradient elution) before testing on gels, or you should do a spin dialysis to concentrate after gelfiltration before running the gel.
Hi Jurgen
Many thanks for your reply.
I am purifying protein from culturables without any tag so many steps are required for its purification as with one step many proteins come together due to same properties.
Ion exchange is my first step and Gel filtration is my last step.I also tried to scale up the initial volume but at the end when i have to inject the protein i concentrate the protein to certain concentration as at high concentration it becomes too viscous to be injected in column and then i concentrate the fractions that show activity and after that i run gel even then nothing is seen on sds gel.
what do you suggest ??
Have you measured the protein concentration in the purified sample? How much was loaded onto the gel?
What is the composition of the sample buffer? What I'm wondering is whether there are substances in the sample buffer present at high enough concentration to interfere with the protein running on the gel. (For example, if there is a lot of a non-ionic detergent in a sample, it may prevent the proteins from running into the gel.) Since you mentioned that the sample injected onto the GFC columns was quite viscous, it occurred to me that there may be something in there in addition to protein.
I loaded 15 -20ug of protein
sample buffer has SDS, b mercaptoethanol,bromo phenol blue,glycerol and tris cl buffer ph 6.8
Salt conc is also less when sample is injected in GFC column. I don't understand what is actually happening as activity of protein is increasing but I am not able to check its quality on gel
15-20 microgram of total protein should be detectable even with Coomassie stain, unless it is a very complex mixture of low abundant proteins of which your protein of interest is just one. Your sample buffer is slightly acidic, which is unusual for denaturing gels. pH 6.8 is the pH of the stacking gel, the whole idea of this is to use alkaline samples so that the proteins have strong negative charge. You should try our protocol:
Sample buffer (keep at 4 degrees):
0.1 % Bromophenol blue, 5 mM EDTA, 200 mM Tris pH 8.8 , 1 M Sucrose
"Sample buffer mix" (always make fresh)
900 ml Sample buffer, 300 ml 10 % SDS, 20 microliter 1 M DTT (from frozen aliquots)
Our stacking gel contains 20% sucrose rather than glycerol, otherwise it is standard PAGE, stacking gel pH 6.8, separating gel at pH8.8.
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