Biotin-sreptavidin binding - (Sep/21/2006 )

Hello
I have a question...
How I can check if the biotin is binding the streptavidin ???

Can I check it in a SDS electroforesis ? (I try it in a native gel but I can't see nothing)

Please... if someone can help me with these...
Thanks a lot

Kiklio

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I would better do it in a native gel.
if you do a SDS-PAGE you will separate your biotinylated protein and streptavidin.

load the biotinylated protein in one lane, and the biotinylated protein incubated with streptavidin in the second lane. in the second lane you will have a big aggregate that will not enter the gel, and you won't see it.

so, if your protein "disappear" it means that the streptavidin was bound.


did you have a control without streptavidin?

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Are you doing an ELISA, western, or IHC?

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What about Pierce HABA calculator? although recommended for biotin-avidin, it should also work for streptavidin; it is a simple quantification assay for avidin-biotin binding with the avidin-binding dye HABA; competition with your biotin decreases HABA absorbance at 500 nm

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It may not be sensitive enough, therefore requires quite bid of samples.

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the advantage of the assay on a native gel is that you visualise very quickly the amount of protein that is biotinylated.
In fact, you see if there is still some non biotinylated proteins, because the migration of the streptavidin biotinylated protein complex is shifted.

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