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problem: doublets and triplets of bands occur in SDS-PAGE


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#1 haiying

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Posted 11 November 2009 - 09:34 PM

Hi, all,

I expressed a fusion protein with mbp tag (MW: 81KDa+42KDa) and everything looks fine. Molecular weight from SDS-PAGE matches well with above. After the cleavage, it shows clearly a single band at 81 KDa and mbp single band (42KDa). After size exclusion column, I got the pure target protein (81KDa). However, from SDS-PAGE gel (16% + 5%), I got several discontinuous bands around 81KDa (they are really very close to each other). I know my protein is pure. I don't know the reason.
I made fresh APS solution and didn't make any difference. I tried to load different amount of proteins and the single bands occurs for smallest amount of samples.

The same situation happens for my another protein which has the molecular weight of 54KDa. But I haven't had the same problem before. Is there anyone who can give me some suggestions.


Thanks.

Haiying

#2 Prep!

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Posted 11 November 2009 - 09:48 PM

if it was not happenign before and happening now.. i see only 2 areasd weer it must be affecting.. one is ur SEC... check the buffers used.. column resin etc (may have been degreaded wrt to ur previous times) and the SDS buffers.. even SDS matters if it is too old... specially in the sample buffer.. i presume u are doing a non reduced gel... try a reduced and see the difference.. but first trouble shoot the error in ur SEC step or the SDS gel stock solutions..
hope this helps...
usually sec does not change the 3 d structure but if the matrix is corrupt or the buffers are... this may happen!!
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#3 mdfenko

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Posted 13 November 2009 - 11:29 AM

you may be seeing mild proteolysis.

how fresh is your gel filtration column?

for what else has it been used?

we found that a gf column retained some proteases from a previous procedure and they partially degraded a protein we ran later. maybe this is happening to your protein.

have you compared the band size of the post gf protein to that of the original cleavage product?

have you determined that the enzyme you use to cleave the mbp from your protein has no effect on your protein? prolonged exposure may have allowed some proteolysis.
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#4 haiying

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Posted 14 November 2009 - 11:38 AM

Thank you guys. I think buffer is fine for the protein and I use it from the beginning of the purification. The enzyme we use for cleavage is TEV, which doesn't cleave my protein I checked.

It might be the SEC column where my protein is degraded. What should I do to check it? Wash it carefully. Do you think if I add some protease inhibitor or PMSF to my protein solution before I run the column, it will be helpful?

#5 mdfenko

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Posted 18 November 2009 - 01:21 PM

Thank you guys. I think buffer is fine for the protein and I use it from the beginning of the purification. The enzyme we use for cleavage is TEV, which doesn't cleave my protein I checked.

It might be the SEC column where my protein is degraded. What should I do to check it? Wash it carefully. Do you think if I add some protease inhibitor or PMSF to my protein solution before I run the column, it will be helpful?


you could add inhibitors to your buffer.

you could clean the column with naoh (see the cleaning procedures that come with the column).
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#6 Feelcontraire

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Posted 18 November 2009 - 04:38 PM

Hi, all,

I expressed a fusion protein with mbp tag (MW: 81KDa+42KDa) and everything looks fine. Molecular weight from SDS-PAGE matches well with above. After the cleavage, it shows clearly a single band at 81 KDa and mbp single band (42KDa). After size exclusion column, I got the pure target protein (81KDa). However, from SDS-PAGE gel (16% + 5%), I got several discontinuous bands around 81KDa (they are really very close to each other). I know my protein is pure. I don't know the reason.
I made fresh APS solution and didn't make any difference. I tried to load different amount of proteins and the single bands occurs for smallest amount of samples.

The same situation happens for my another protein which has the molecular weight of 54KDa. But I haven't had the same problem before. Is there anyone who can give me some suggestions.


Thanks.

Haiying


I think that you have been very well advised, if it where doublets you would see much distant bands.

The problem seems like mild proteolysis or bad S-S reduction.

If you are sure the column is clean ad protease inhibitors as soon as you collect the sample from the column.

If you have already tried mercaptoethanol and DTT y suggest trying neutral ph TCEP for sample reduction, it is very stable and will keep your samples reduced.

If it came from a deficient gel you would probably see a smear instead of discrete bands.




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