Strange results in biotin-avidin pulldown - has anyone ever got it like that?????? (Apr/26/2011 )

Hi,

I have a very strange question....I am not able to get any answer .....OK...so I have a biotinylated probe that I am using to see in the lysate if some protein binds to it...for this I am using dynabeads which are magnetic beads coated with avidin. I do pulldown experiments and use only biotin as negative control. Now when I run the gel, I don't see any difference in both the lanes(biotinylated probe and biotin). But when I probe the same gel with avidin-HRP antibody I see one band only in the lane containing biotinylated probe and not in biotin containing lane..which is good in a way ...but if the protein is specific to my biotinylated probe then why is it showing up in the gel in both the lanes ......I don't have any explanation for that ......can somebody help me??????

thanks

does yr samples that u apply to the gel are dynabead free ?
Biotin react quick when it is in solution thats why you have to prepare it each time new. so when your samples with the biotin have time to "react" (up to 15min) then you wont see a specific binding to this.

what does it mean " when I run the gel, I don't see any difference in both the lanes"? Did you stain proteins in gel or on blot and if, by which staining method?

Hello,

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 !