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.
Did you equilibrate the resin with lysis buffer before adding it to the sample?
Adam B Shapiro Yes I equilibrated the resin before hand.
Tomáš Hluska Sample was in 500mM NaCl, 10mM TCEP, 20% glycerol, 100mM KPI, 1mM EDTA, 1uM BHT. Resin was in 100mM KPI, 20% glycerol, 500mM NaCl. 0.5mL resin to 200mL lysate.
Ethan Matthews it's funny that you write the composition in different order xD
Anyway, was this your first time or did you perform this extraction previously? What if you mix the buffer in which was your sample (KPi, glycerol, NaCl, TCEP, EDTA, BHT) with the resin in the same ratio?
If BHT is butylated hydroxytoluene, that seems to be lipophillic, so that could become cloudy when diluted in water/high ionic solution, but you have it in the same conditions in your sample (I assume the pH was the same as well), so that shouldn't matter.
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