The supplier COA stated that the snail protein is about 30KD. I ran the electrophoresis at 200V, 125mA for 45mins. I used 4-12% Tris Nu-PAge Gel. Why is the snail protein more than 30KD?
My first couple of questions are:
1. What steps did you take to denature prior to loading?
2. What ladder did you use?
3. What was your running buffer, the buffer in which you run (MOPS, MES, etc.) will result in different ladder-running profiles.
4. How much of a difference in size did you see?
It seems other people see the protein at 29kDa. Either your molecular weight marker is not accurate or your antibody is recognizing a different protein. Post some more details if you want more specific advice. Are you working with human samples? Who is the manufacturer of the antibody? Post a link to the product page.
i have just reviewed my log book. I think i forgot to mix reducing agent (commercial product) together with LDS and protein lysate before I loaded them. Will this affect the protein migration?
a) Step of denaturation prior to sample loading: protein lysate + LDS + reducing agent (i forgot to add this) then proceed to 70 Celsius heating for 15mins
b) material and reagents are all commercial. Antibody should be fine as it is Cell Signaling Snail antibody. Ladder is Precision Kaleidoscope
c) Those protein lysates derived from human cancer cells.
Thanks Joseph and Mark
Non-reducing conditions (as you forgot the reducing agent) can easily affect the protein band in its size.
And even if you have the reducing agent in it, it is often possible to get other band sizes than the expected ones, depending on the type of protein or postranslational modifications...
I think it is the absence of the reducing agent but other possible options could be ubiquitinilation, phosporylations (higher bands) or different isoforms. For example I examine a transcription factor that occurs in 8 different isoforms (depends on cell type) and can be phosphorylated 6 times. Because of this I get several different bands in my WB.
Actually, I have the same problem, that is not a problem at all.
Snail is often found at a different molecular weight because of several post-translational modification . It's difficult to explain but, it's science ;)
And if you check it out (also in antibodies lists of many companies, you will found different antibodies detecting for Snail at different MW)
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