Hi,
I am working with a mesenchymal cell line. My protein of interest is around 57 kDa and there is a published report on this cell line for the same protein. But the problem is, when I do the same western blot, and I use the same antibody as they used in the report, I am not able to get its band in to be the same size and my size is always less than 50. Can anyone explain this behavior of the cell line? What are the possible causes of this.
Well, try to run with a positive control (cell line well known to express that protein). I don't think the size would differ from cell line to cell line, unless other factors like cleavage or other modification process affecting your protein. I haven't come across such problem, but I don't think same antibody would give different band size in different cell lines, unless he protein has been modified. I would suggest to re check how you running the gel. Are you using the right blocking buffer? Make sure the pH is not changing or gel is hot. Check all possibilities that would affect your migration.
Hi Jaya, Emmanuel has made some good suggestions. As to your question "what are the possibility for getting this": If you are using the same antibody (company and catalogue) then the difference must be in the line itself.
There are a number of possibilities:
a) The original publication is incorrect: have you calculated the theoretical size of the protein from it's primary structure? The actual size may be different due to modification by i.e. glycosylation but it is worth checking this. Also check the literature for anyone else detecting the protein on western blot (and see what size they get). There are also programs on-line that can identify possible glycosylation sites, etc.
As Emmanuel suggests if you can obtain a second cell line known to express this protein you can run alongside your cells and see if there are any differences in size (if you use a primary cell such as dermal fibroblasts, ATCC, PCS-201-010 then you can be fairly certain that the protein you are detecting in this line is un-mutated).
b) The protein in your cell line has become truncated/mutated: cells in culture are always under selective pressure. If passaged too long they can gain mutations that give them growth advantage (in the same way cancers adapt). If you have sufficient funds and access to more cells, I would be tempted to purchase a new stock of cells. If you do then expand for a few passages and freeze down a Master bank. Once this has been laid down expand and freeze down multiple working banks. Return to the working banks each time you need to expand sufficient cells for the analysis.
c) The conditions for the western blot are incorrect - especially the loading buffer.
I assume you are using a protein ladder from a commercial source (i.e. the ladder is not in the same loading buffer as your sample). This means that if there is something wrong with your loading buffer (i.e. too much salt is present in it or you are adding too much sample (overloading)) your protein may run aberrantly. This is unlikely as when this happens your target protein tends to run at a higher MW than it actually is (the opposite of what you are seeing).
d) The protein you are looking at is modified in culture. Check that the original article is cultivating the cells at the same density, in the same media and with the same growth factors. It may be possible that the cells are responding to something that is cleaving the protein (or modifying it in the original article).
You could try obtaining a second antibody to the protein too (if your ab recognises N-terminus get a C-Terminus and vice-versa). This is just in case that the epitope your ab recognises has been masked somehow and you are detecting a related protein (this is unlikely however - but you can look at the original blot that places the target at 57 kDa and see if there is a secondary band at the size of your protein in the auto-radiograph.
I mentioned in b) above that you protein may be truncated - but perhaps this is happening due to cleavage rather than mutation so check if you are including the same protease inhibitors in the lysis buffer etc that the original work uses.
The only other thing I would suggest trying is to PCR up the exons of the gene you are interested in (either by RT to get the entire coding region or from the genomic) and sequence several clones (I would suggest multiple clones as PCR errors can occur). This would allow you to check the sequence to see if there are any mutations that are shifting the coding region out of frame or introducing stop codons.
Tracking down the cause of weird results is unfortunately "par for the course" in science. I hope this helps!
Gary
This could be a problem of cleavage or ubiquitinylation of your core protein.
Thank you all for the suggestions. Probably I will try with a new stock and expect the result would be good
1. This could be just due to the differences in percentage acrylamide used to separate proteins. SDS-PAGE is not an exact science and is semi quantitative. Also the use of different standards.
2. It could also be due to the way the cells have been grown and the way the sample has been handled post expression: media differences, cell viability, general health of the cells. This can cause differences in gycosylation and/or amiino acid modifications/cleavage of protein. Use protease inhibitors if necessary.
3. Were samples reduced or non-reduced when analaysed: this will show up differences due to disulphide bonds.
good luck
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