Hello everyone,
To increase the binding of my StrepII tagged protein to the Strep-Tactin®XT 4Flow high capacity resin, I have been incubating the resin with my lysate before adding to a column. I noticed when I add the resin the lysate becomes rather cloudy. I tried to add it to the column and it was completely clogged, no dripping at all. I had to complete the rest of the purification using a batch purification protocol which gives me lower yield and purity.
Sample was in 500mM NaCl, 10mM TCEP, 20% glycerol, 100mM KPI, 1mM EDTA, 1uM BHT. Resin was equilibrated in the same buffer. 0.5mL resin to 200mL lysate.
I've experienced similar issues using Ni-NTA resin and recently scanning researchgate for solutions.
The precipitate that forms is protein aggregation, is it not? So maybe working in a cold room is best?
I've completely given up on incubating resin with cell lysate. Even after short incubation you will see strings of snot form and then it is impossible to filter out your resin from precipitation. Clarify lysate by centrifuge, then by vacuum filter, then add right to column. If the product manual indicates that low concentrations of detergent are tolerated, small amounts of SDS can help. Very low conc. won't damage enzymes, will aid in lysis and foaming shouldn't be an issue if
James Carrillo I'm not sure what it is but my guess would be that it is some weird type of protein precipitation. It only happens ~1 hour after I add my resin and stir. I ultracentrifuge my lysate at 35Krpm for 30min and always work in the cold room. I just dont understand why its happening and how to prevent it.
Perhaps it is DNA and/or RNA. Have you tried adding DNase + Mg2+ and RNase?
Since you said you are observing this 1 hour after the addition of the Strep-Tactin®XT 4Flow...Did you check if this is also occurring at the end of the 1 h incubation without the resin at cold conditions? Just incubate the cell lysate at cold conditions without resin to simply elucidate if the problem is only arising when the resin is existence...
If yes and if this is not a kind of aggregation/precipitation problem (you may test by analyzing the precipitates), we may think that the solubility of something in your lysate is dependent on the tagged protein (carrier) which you retain on resin as batch mode, and losing its carrier molecule in time resulting reduced solubility.
James Carrillo I think I figured it out. I tried doubling my lysis buffer, doing the binding at room temp and adding 0.06% Octyl β-D-glucopyranoside to prevent my protein from aggravating. It worked!
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