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Protein Purification from Polyacrylamide Gels

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#1 PancakeSorting



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Posted 14 January 2013 - 11:28 AM

Hi everyone,

Recently I've been attempting to purify a GST tagged protein. It's a pretty small protein, ~20 kd, but with the GST tag it's about ~45 kd total. This is recombinant mouse protein produced in E. coli. I get a decent yield of protein from the E. coli, but the E. coli don't seem to particularly like making my protein, so they'll often stop making it at certain points in the sequence, leaving undesirable shortened forms of my protein floating around in my final solution (I should add that these shortened bits appear after purifying with glutathione, bc the GST tag is on the N term so all of the shortened bits have the GST tag and thus are pulled out of solution along with the full version bits). There are also a few higher MW things in the solution as well, possibly dimers/trimers. So I thought I'd try to purify my protein using a nuPAGE gel purification approach, since it seemed quite simple, but it isn't working and I'm not sure why.

I followed this protocol: (see attached for full protocol if you're interested)
Run gel with protein (~1 hour at 200 v)
Excise desired band (stained side strip of gel with Coomassie blue and then cut out where band of interest should be)
Chop up gel into bits
Use pestle to grind up gel bits
Add ~1 mL buffer ( 50 mM Tris-HCl, 150 mM NaCl, and 0.1 mM EDTA; pH 7.5)
Leave overnight at ~30 degrees C to allow for passive transfer
Centrifuge and collect supernatant

But when I run a sample of my supernatant on a gel, there's no protein. I'm puzzled as why this isn't working. I double checked my buffer and even allowed for 2-3 days at 30 degrees C and I've stained the remaining portion of my gel to ensure that I wasn't accidentally leaving my desired band behind.

The only thing I can think of is that this buffer isn't appropriate for my gel/protein or I'm not achieving sufficient gel disruption to allow the protein to leave the gel.

Any ideas? I don't have access to dialysis or electrocution equipment. I've been thinking of maybe sonicating my gel mix. But would another buffer maybe work better? I'm using a nuPAGe 4-12% BisTris gel 1.5 mm btw.

I just really want to purify this protein so I can run some assays on it but I work in an Immunology lab so I don't have access to typical proteomics equipment. Blah.

Any help/advice would be much appreciated!

Attached Files

Edited by PancakeSorting, 14 January 2013 - 11:37 AM.




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Posted 15 January 2013 - 01:22 PM

Is it possible that the protein precipitated and it is being trapped in the pellet after centrifugation? In this case, you can optimize the buffer to reduce precipitation and your protein will stay in solution in the supernatant. You could try using a buffer with HEPES, 5-10mM DTT, Glycerol. Maybe tris is the problem?

#3 PancakeSorting



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Posted 16 January 2013 - 11:12 PM

I hadn't thought of it precipitating out, I'll give HEPES a try and see what happens. I'll update with the results. Thanks for the suggestion!

#4 Adrian K

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Posted 17 January 2013 - 07:25 AM

Another alternative: try electro elution method.
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