Hi All,
I would like to get some feedback about people's experiences when labeling a his-tagged protein with anti-his antibody. What proportion of protein to antibody is suitable? I have very little protein, ~10 uL of ~7 uM protein. The antibody I am using is 125 μl Penta·His Biotin Conjugate, 200 μg/ml from qiagen.
Also, what are the conditions for incubation? Are there any good references I could read?
Thank you,
Monika
Hi
I used the Penta·His Biotin antibody diluted at 1:1000, incubation time 1.5h at RT. At the beginning, you can use different dilutions 1:500, 1:1000 and 1:2000. Conditions are dependent on your protein.
What protein concentration did you use for the 1:1000 dilution?
Thank you.
Hi
I used ~ 50 nM protein concentration. in other words, for detection I used 150 ng of bacterial lysate containing His-tagged protein or 15-20 ng of purified His-tagged protein.