Biotin- avidin pulldown assays - basics (Sep/24/2010 )

Hi,

Biotin-avidin pulldown assays are very common and one can find lot of papers ...but still I need help in this . I am getting lot of nonspecific binding . my protocol is very simple, I have to elute biotinylated proteins from lysate, using avidin agarose. I incubate lysate with avidin at 4 degrees for an hour, spin it down, throw the suppernatant, wash the beads twice with phosphate buffer pH-7,and finally add SDS dye, in the beads, heat it and load on the gel. I am taking negative control also, but problem is, i am gettin proteins in both negative control and my sample .....I hate it ...has anybody used dynabeads(magnetic strptavidin beads)..hows the experience?...help me please, since no one around does this kind of work, i don't have anybody to ask, i've tried to read papers, but no help .....

thanks

am2

it is a method to enrich f.i. surface proteins; an unspecific binding to beads and avidin is given and not unusual; you may take more washing steps but may lose more protein...

are you using salt in the wash buffer?

you need to maintain an ionic strength of at least 0.15M (or 0.2M, depending on the matrix) to prevent (or reduce) non-specific binding to the beads.

Thanks for ur suggestions. yes I was not usng salt in my washing buffer, i was using just phosphate buffer to wash the beads.I will try using salt in the buffer, hope that will give me some positive results

I used dynabeads magnetic beads and they work quite well. I would wash more times and maybe try a elution from the beads with biotin (competition), you will have less unespecific proteins in your elution. You can try as well a mild elution with Citric acid ( I am not sure about that, because it has been sometimes since I used them, but you can check in the datasheet)

Try adding Tween-20 or Triton x-100 (.2% - 1%) to the wash buffer along with 0.1%SDS. Wash beads atleast 4 times. Also maintain 150 mM NaCL. You could try upto 500mM. But I would first try with Tween-20/Triton with SDS.