protein band is shifted after boiling sample with sample buffer - (Jul/02/2008 )
Hi, All
My target protein is 100KD, monomer receptor. I used regular Laemmli sample buffer before running SDS PAGE. Without boiling sample it showed a strong band at 100KD. But when the sample was boiled at 65 and 95 degree, the 100KD band disapeared, instead of there is a 50KD band showing up. Does that mean the protein is broken down into two pieces of 50KD protein? Can boiling cause protein degradation?
Boiling in denaturing buffer should result in the protein being completely denatured, including any dimers etc that may be there. Seeing as your target protein is 100 kDa as a monomer, there should be only one band somewhere near 100 kDa. Having said that, there are a number of factors that can affect how the protein runs on the denaturing gel, the biggest of which is the number of charged amino acids in the protein, which may affect the running size by quite a bit (I'm working on a protein that has a MW of 37 kDa and it runs at 50).
I would suggest checking the antibody data sheet to see if they have the running size, it may be that you used the wrong antibody, or that the literature is wrong about the protein being a monomer.
I would suggest checking the antibody data sheet to see if they have the running size, it may be that you used the wrong antibody, or that the literature is wrong about the protein being a monomer.
Thank you.
The antibody was correct and the MW of protein is 100KD based on amino acid sequence. I did the experiments at the same time, the same dilution of the same antibody. The only difference was boiling and non-boiling from the same cell lysate. Without boiling everything is fine. I just can not figure out why the boiling caused the running size becoming so small (50KD). I also see dimer at around 200KD without boiling. After boiling I do not see monomer and dimer, but only see 50KD. I am so confused.
Please someone helps me.
My target protein is 100KD, monomer receptor. I used regular Laemmli sample buffer before running SDS PAGE. Without boiling sample it showed a strong band at 100KD. But when the sample was boiled at 65 and 95 degree, the 100KD band disapeared, instead of there is a 50KD band showing up. Does that mean the protein is broken down into two pieces of 50KD protein? Can boiling cause protein degradation?
Question:
If I understand you correctly, you're saying that the SAME antibody detects the presence of the protein but the protein harvested without boiling shows up at 100KD and the protein harvested using boiling shows up at 50 KD...
Is that correct?
If so, and given that your antibody isn't likely to lie to you, it seems logical to assume that the boiling, for whatever reason, does lead to a cleavage of the protein.
Think on it. The antibody has a specific binding epitope and that epitope is found on the protein you are investigating.
There are ways, however, to be cetain...
1: repeat your protocol, but in one sample (prior to boiling) add a known amount of the protein you are investigating. You'll know it's there, you know it's weight. If it shows up at 50 KD then you can be more certain that the boiling is causing cleavage.
I would also suggest you make sure that the protein is not running faster which could cause the 100 KD protein to run off the gel and not appear at all... By this i'm not suggesting that cleavage is not occuring, but I would expect SOME of the 100 KD form of the protein to still exist. If it is no longer showing up either it is gone before the gel is run, or it is running off the gel.
I hope this helps.
My target protein is 100KD, monomer receptor. I used regular Laemmli sample buffer before running SDS PAGE. Without boiling sample it showed a strong band at 100KD. But when the sample was boiled at 65 and 95 degree, the 100KD band disapeared, instead of there is a 50KD band showing up. Does that mean the protein is broken down into two pieces of 50KD protein? Can boiling cause protein degradation?
Question:
If I understand you correctly, you're saying that the SAME antibody detects the presence of the protein but the protein harvested without boiling shows up at 100KD and the protein harvested using boiling shows up at 50 KD...
Is that correct?
If so, and given that your antibody isn't likely to lie to you, it seems logical to assume that the boiling, for whatever reason, does lead to a cleavage of the protein.
Think on it. The antibody has a specific binding epitope and that epitope is found on the protein you are investigating.
There are ways, however, to be cetain...
1: repeat your protocol, but in one sample (prior to boiling) add a known amount of the protein you are investigating. You'll know it's there, you know it's weight. If it shows up at 50 KD then you can be more certain that the boiling is causing cleavage.
I would also suggest you make sure that the protein is not running faster which could cause the 100 KD protein to run off the gel and not appear at all... By this i'm not suggesting that cleavage is not occuring, but I would expect SOME of the 100 KD form of the protein to still exist. If it is no longer showing up either it is gone before the gel is run, or it is running off the gel.
I hope this helps.
Thank you so much for your suggestion. It is a very good idea to add a known protein. I wish I have the protein.
I have repeated the protocol for 3 times and my co-workers also tried. We all got the same results. We try to detect the receptor changes in samples from the rat brains. We tried the regular western blot protocol with boiling prcedure at the beginning but did not get the band at the right size (100KD), we only see 50KD. Then we changed it into non-boiling protocol, and the band showed up right on the target size. We've never seen this with other proteins before. To my knowledge the boiling procedure itself will not cleave protein. Am I right? Or the 5min 95 degree boiling does cleave the protein? Please correct me.
Thank you for your help.
You're correct in that the boiling is not SUPPOSED to cleave the protein, but in your case nature seems to be willing to make an exception. 
Remember, there are very few (if any) rules in biology that are absolute.
I think you've found something interesting. Can you tell me what receptor you are investigating?
Tim
Remember, there are very few (if any) rules in biology that are absolute.I think you've found something interesting. Can you tell me what receptor you are investigating?
Tim
Of course. It is mGlur1B. It is a very abundant receptor in the brain. I m sure you know more than I do. Maybe I will ask you more questions later on.
Thank you for answering my question. I greatly appreciate your help.
Have a nice day!
deventx - "I m sure you know more than I do. "
I so wish that were true, but I doubt it...
Once in a while we end up with data that isn't what we expected it to be, most of the times it doesn't make sense to us. Our first reaction is ussually to expect that we've made some sort of experimental error and need to re-run the experiment...
But after being certain about the protocol, and in the absence of any other information...I think it's fair to trust the data...
Here, you've rerun your protocol several times. I believe you said, others in your lab got the same result?
Well, given the specificity of primary antibodies, I think you can trust your data. You may not know the exact mechanism, but you do have clear evidence that the protein is being cleaved.
The only other thing that comes to mind is using a different primary antibody to ensure that you get the same results. If both antibodies show the presence of the protein at 50 KD after boiling then you have solid data that suggests the protein has been cleaved...
you could be onto something interesting