band running at wrong(?) size! - more than one band, and at almost twice the predicted size. (Jun/27/2007 )
Hi everyone.
I am trying to run a western for a protein that has a predicted size of ~70Kda. I get more than one band, at ~140Kda.
I use 1%SDS in my sample buffer, and 1%SDS in my running buffer. I also boil my sample (mixed with the sample buffer) for ~4-5 min before I load it on the gel.
I also see a very very faint band at 70Kda if I load a LOT of protein in the wells.
1) What could be the possible reasons, I am heating it enough, and using SDS, so technically there shouldnt be any dimer's I guess... can dimers exist even after the treatment I give them? Could using urea help in any way??
2) Also, I am not sure about the post-transcriptional modifications on the protein! I looked up EXpasy and there are some predicted phospho and glycosylation sites, but does any one know of some tool to help predict how much the mol wt could be expected to increase after the predicted modifications? Is there any other better tool to search for potential modifications?
I have the immunizing peptide, but blocking isnt consistent, I have tried it more than twice, and I did not get consistent results.
I will also look at other topics in the forum and in the Bioinfo forum to see if these issues have been discussed before. But any suggestions/help is be greatly appreciated!
Thanks,
Vik.
I am trying to run a western for a protein that has a predicted size of ~70Kda. I get more than one band, at ~140Kda.
I use 1%SDS in my sample buffer, and 1%SDS in my running buffer. I also boil my sample (mixed with the sample buffer) for ~4-5 min before I load it on the gel.
I also see a very very faint band at 70Kda if I load a LOT of protein in the wells.
1) What could be the possible reasons, I am heating it enough, and using SDS, so technically there shouldnt be any dimer's I guess... can dimers exist even after the treatment I give them? Could using urea help in any way??
2) Also, I am not sure about the post-transcriptional modifications on the protein! I looked up EXpasy and there are some predicted phospho and glycosylation sites, but does any one know of some tool to help predict how much the mol wt could be expected to increase after the predicted modifications? Is there any other better tool to search for potential modifications?
I have the immunizing peptide, but blocking isnt consistent, I have tried it more than twice, and I did not get consistent results.
I will also look at other topics in the forum and in the Bioinfo forum to see if these issues have been discussed before. But any suggestions/help is be greatly appreciated!
Thanks,
Vik.
there is too much heating; do not heat more than 1 min, or better try 15 min at 60°C as there seem to be aggregations of your p70;
PTM to add >70 kDa may not happen
I am trying to run a western for a protein that has a predicted size of ~70Kda. I get more than one band, at ~140Kda.
I use 1%SDS in my sample buffer, and 1%SDS in my running buffer. I also boil my sample (mixed with the sample buffer) for ~4-5 min before I load it on the gel.
I also see a very very faint band at 70Kda if I load a LOT of protein in the wells.
1) What could be the possible reasons, I am heating it enough, and using SDS, so technically there shouldnt be any dimer's I guess... can dimers exist even after the treatment I give them? Could using urea help in any way??
2) Also, I am not sure about the post-transcriptional modifications on the protein! I looked up EXpasy and there are some predicted phospho and glycosylation sites, but does any one know of some tool to help predict how much the mol wt could be expected to increase after the predicted modifications? Is there any other better tool to search for potential modifications?
I have the immunizing peptide, but blocking isnt consistent, I have tried it more than twice, and I did not get consistent results.
I will also look at other topics in the forum and in the Bioinfo forum to see if these issues have been discussed before. But any suggestions/help is be greatly appreciated!
Thanks,
Vik.
hi vik..
I am sorry .. but i never see any relationship between SDS and dimerisation.. you are confused between B-Mercaptoethanol and SDS.
People use B-Mercaptoethanol to test ...............if thier protein is covalent dimer or not....... B-Mercaptoethanol reduces any disulfide bond..
therefore people run two gels.. reducing(B-Mercaptoethanol) and non-reducing (without B-Mercaptoethanol) to thest protein aggregation or dimerisation..
you should run both gels.. u'll know whats going on..
n hann.. somtimes proteins are non-covalent dimers... so u can't rule that out..
forget about other stuff like sugars and post-transcriptional modifications...#
check this paper.. to see both blots..
http://journals.iucr.org/d/issues/2002/04/.../gr2205bdy.html
Okay, the most likely problem here is that your antibody is crap.
I know the answer before I even ask but did you run cell extracts
that serve as a positive and negative control?
I have used antibodies especially from Santa cruz and detect non-specifc
high molecular weight crap.
The only way to sort it out is to A) Use positive and negative controls.
Transfect cDNA encoding your protein and see if the antibody detects it when overexpressed.
As a general rule, when something doesn't work in Western Blotting the answer isn't complex.
Thank you all for your replies. I guess no-one thinks that any PTM might be an issue!!
I still have a few more questions...
1). I do use BetaME in my sample buffer, but are there any interactions that are not broken by BME. Would using a denaturing gel (urea?) be any use in that case?
2). I am using whole serum from the immunized rabbits, could that be a cause for the interference?! I might have some pre-immune serum as well, and I will try running that, but could non-purified antibodies cause such high levels of non-specific binding, assuming it is non-specific!
3). There are no mutants available for the protein that I am looking for, so I cannot really run a negative control!! alllthough the pre-immne serum could be considered one!
4). Incidently though, the peptide (we had the peptide synthesised and antibodies raised commercially by Covance!!) has an ~10amino-acids align with another protein of about 150Kda!! Could it be possible that my antibody is actually recognizing this other protein better than the target protein!!!, in which case, I guess the only option remaining is to express the protein in a cell-line that does not normally produce either of the proteins!!??
thank you in advance for any suggestions!!
Vik.
and oh yes... I AM using direct tissue, homogenized in protein extraction buffer as my samples!!
Hi again,
yes it is possible that your antibody is cross reacting with a similar protein.
By a negative control I mean use extract from a cell line that does not express your protein.
If you are studying a new protein do RT-PCR on a number of cell lines and see if it is
silenced in a particular cell line.
It is possible that your protein runs anomalously as a high molecular weight protein.
In vitro translation of your protein followed by Western blotting or S35 label your in vitro translated product
could also help sort this out.
Also, again, overexpressing your protein in a cell will also help because you should get a big "blob" when Western blotting these cells.
Good luck!