Biotin-strptavidin pulldown experiments
Posted 25 January 2011 - 07:02 AM
I have posted a question about this earlier also..but still struggling for answers . I am doing biotin-streptavidin experiments on cell lysate..looking for a protein that binds to my biotinylated probe. my problem is that I am getting lot of nonspecific binding which is very normal in this case, to get rid of this problem, I used 0.1% SDS in washing buffer..now the nonspecific binding has reduced, but I am worried..what if this SDS releases the specific protein also in washes ....I read about biotin-streptavidin interaction..I know that is quite strong and SDS can't do anything to break that interaction...but I don't know how strong is the binding between my probe and the protein..so when I add SDS..ideally it will act on everything ...I don't know whether I am right ......what to do .....does anyone has some better protocol for this..which can be shared??? ...and one basic question..people use DTT also in the washing buffers..I know its used for the reduction reaction but why in this case?? how does it help to reduce nonspecific binding..I tried to search on the net but couldn't find the reason ......please please someone please help me
Posted 04 August 2014 - 03:13 AM
I've made a little research about streptavidin-coated magnetic beads. I would like to immobilize biotinylated PCR products. I have 3 candidate products. Theoretically, all of these items are ideal for this purpose.
There is huge price difference between these products (and some differences in certain parameters).
- Dynabeads® M-280 Streptavidin ; bead diameter: 2,8 um ; Binding Capacity (free biotin): 650-900 pmoles/mg beads ; concentration: 10 mg/ml ; 10 ml : 1 500 $
- solulink ; bead diameter: 0,8 um ; Binding Capacity (free biotin): > 14 nmol/mg ; concentration: 10 mg/ml ; 10 ml: 800 $
- spherotech ; bead diameter: 0,5 - 4.5 um ; Binding Capacity (free biotin): no described ; concentration: 0,5 - 1 w/v % ; 10 ml: 200-250 $
But I don't know what so special in M-280 making it very expensive.
Can anybody tell me what I should choose ?
Thanks for the answer !