precipitation during TEV cleavage - (Oct/21/2008 )
Hello,
I notice that my MBP-tagged protein complex gives heavy precipitation during TEV cleavage to remove the tag. I've run a gel on the precipitate and confirm that it is TEV, MBP, as well as my protein complex. Prior to TEV cleavage, the protein was purified by nickel affinity chromatography (His tagged on a different subunit). I believe it is not aggregated at the time of nickel affinity purification because the whole complex is eluted while only one subunit has the His tag. My TEV cleavage conditions are typically over night at 4 C, 0.5 M NaCl, 1 mM MgCl2, 10% glycerol, 0.5 mM TCEP. I'm usually working with 10-50 mg protein in about 15 mL buffer.
Thank you!
-hln-
QUOTE (hln @ Oct 22 2008, 04:22 AM)
Hello,
I notice that my MBP-tagged protein complex gives heavy precipitation during TEV cleavage to remove the tag. I've run a gel on the precipitate and confirm that it is TEV, MBP, as well as my protein complex. Prior to TEV cleavage, the protein was purified by nickel affinity chromatography (His tagged on a different subunit). I believe it is not aggregated at the time of nickel affinity purification because the whole complex is eluted while only one subunit has the His tag. My TEV cleavage conditions are typically over night at 4 C, 0.5 M NaCl, 1 mM MgCl2, 10% glycerol, 0.5 mM TCEP. I'm usually working with 10-50 mg protein in about 15 mL buffer.
Thank you!
I notice that my MBP-tagged protein complex gives heavy precipitation during TEV cleavage to remove the tag. I've run a gel on the precipitate and confirm that it is TEV, MBP, as well as my protein complex. Prior to TEV cleavage, the protein was purified by nickel affinity chromatography (His tagged on a different subunit). I believe it is not aggregated at the time of nickel affinity purification because the whole complex is eluted while only one subunit has the His tag. My TEV cleavage conditions are typically over night at 4 C, 0.5 M NaCl, 1 mM MgCl2, 10% glycerol, 0.5 mM TCEP. I'm usually working with 10-50 mg protein in about 15 mL buffer.
Thank you!
Hi,
The first thing I reacted to is that your protein concentration is very high! Maybe working at lower concentrations can help. Otherwise MBP is known to stabilize proteins that otherwise are not soluble. Cleaving it off could cause precipitation. Your buffer could also be an issue. I assume you elute with imidazole from the nickel column and then change buffer to the cleavage buffer? Maybe the protein is happier in the elution buffer than the buffer you cleave in.
I hope this helps..
-esmeralda-