Do High Molecular Weight Proteins Break Apart In Protein Gels If Run Too Long?

[BNSN]

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Hi, all.

Just wanted some input on this problem I am having:

I am trying to western blot a protein -- Shank -- using an antibody from Santa Cruz. I know the antibody works and gives clean bands at the right size (~130 and ~170 kDA) because a friend of mine in another lab uses it.

My blots, however, give me one and only one band at 75 kDA. Is it possible that this is the Shank protein and that the band represents a physically disrupted Shank protein?

Shank proteins are large and I run my gels at 200 V for 40 mins, which is intense. Could this break up large proteins?

 

[reine1jb]

that is an interesting problem. I don't know anything about the shank protein but a few things I would consider when comparing your protein to your friends results would be:

1) is your protein isolation technique the same or similar, perhaps the protein is getting degraded in your prep...

2) are you looking at shank in the same tissue/cell? Perhaps if you are not, the difference in size could be due to alternative splicing...

3) do you run a lot of westerns? Do you have a control protein that you look at, if you don't, maybe you should to see if that control protein is affected somehow as well, maybe there is something wrong with the gel. Again, I don't know what system you are using but something to think about.

4) I have never run a western at a voltage higher than 150V but again different systems different protocols.

 

[StIGMA]

2 ideas in addition to reine's: Some proteins can degrade if you boil them in Laemmli buffer too long (happens to 2 proteins I work with... but a single product does not accumulate). Cut down your boiling step to <3 minutes. Also, some proteases are activated in the presence of SDS (not sure how common this is). Having the protein break while running on the gel would be at the bottom of my list of possibilities. Do you have any evidence you are starting with full length proteins?

 

[swingline]

Do you use protease inhibitors, such as PMSF or Roche's Complete Protease Inhibitor Cocktail, in your lysis buffer?

PMSF: Prepare 250 mM PMSF in ethanol. Bring the final concentration of the lysis buffer to 0.5 mM PMSF.

Roche's Complete Protease Inhibitor Cocktail: Just add one tablet to the lysis buffer.

I use these protocols for E. coli.

 

[FSAP]

Try 100V for longer periods. It's highly unlikely that protein got degraded in the gel (never heard of if and I am in hardcore proteomics lab). Even if it did happen, I think it will be much more random unless specific covalent bonds are less stable for some reason.

It's much more likely that protein is degraded in the cell and you are seeing its main degraded form. Get the positive control and check if the band is still present there too.

Did you try higher substrate concentrations or hit it with something like femto? Even if you get mad background, if you see a band at high MW, you know your protein is there.

From previous experience 60-75 is where I get a lot of my non-specific bands for no reason. If you are IPing, it's likely IGG and if you are running plasma samples, that's around where albumin shows up.

Another thing I would do is let the gel run longer and allow LMW standards to leak out up until that 60-75 range. That way you will only have your HMW range left. I had problems with some HMW proteins before and that's proven to work.

Take it for what it's worth, but I hope it helps. I am just a lab tech though.

 

[fairalexfield]

A positive control from your friend in the other lab will help immensely.