2 replies to this topic

[neuron]

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

[lab-newbie01]

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.

[Inmost sun]

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?