Who have ever used the streptavidin coated magnetic beads from invitrogen

Hi,now I'm using the streptavdin coated magnetic beads (cat.65205,11205D) from invitrogen in order to capture the biotinylated protein in the solution. But it is very strange,that the bead almost can not capture it.

I have tried so many kinds of biotinylated protein (I did not biotinylate the protein by myself, I bought it from invitrogen,for example biotinylated anti-human IgG which cat.no.H10015), Seems the beads can't capture any kinds of biotinated protein (I follow their protocol strictly,using PBS ph=7.4 as buffer and add 0.1%Tween 20).

I also synthesized some biotinated ssDNA sequences, seem good, the beads can capture the ssDNA.

Who has the experience of using this beads to capture the protein.

You see, after incubation I boil the beads and run a page gel to see if my target protein is on the beads,but there are nothing.

are you including protease inhibitor cocktail in your buffers?

I don't know what is that.My buffer is just PBS(ph=7.4),but I add Tween 20 about 0.1% in the PBS.

 These Complete protease inhibitor tablets are almost a standard ingredient in most protein preparations when you are isolating a protein from membranes or any lysate.

http://www.roche.com/products/product-details.htm?type=product&id=104

without some sort of protease inhibitor in your solutions during the protocol chances are a lot of your sample may be degrading.

Also try not boiling the samples before you run them on the PAGE gel

here is supplemental material from a paper that performs surface biotinylation of membrane proteins.  

http://jgp.rupress.org/content/suppl/2009/12/01/jgp.200910314.DC1/JGP_200910314_sm.pdf

you may find it useful for recipes for your solutions

I think I have to boil the sample.Because the binding of biotin and streptavidin will be released in that way.And my sample is purified protein, not tissue or cell lysate.

 Do you not have Beta mercaptoethanol and/or DTT in your sample buffer to reduce the samples?

Hi There

I am Kristina with Technical Support for Invitrogen Dynal and I'm happy to help with any Dynabeads related questions.

I'm sorry to see that you are having problems with the streptavidin beads, and I hope we can figure out what the problem might  be and get the binging working.

First of all it is important to calcualte the amount of beads needed:

Dynabeads M-280 Streptavidin are supplied at a 10 mg/ml concentration.
One mg og these beads will bind up to 10 µg of biotinylated antibody.
In order to achieve maximum binding capacity it is recommended to use  up to 2-fold excess of antibody.

Binding example:
Take out 100 µl of beads (1 mg), place on magnet and remove supernatant. Wash once in PBS, remove supernatant.
Use 10-20 µg of antibody - dilute this to 100 µl in PBS. Add this to the beads and resuspend.
Incubate the sample for 30 min at room temperature with constant tilting and rotation. -Make sure the beads are fully resuspended at all times throughout the incubation.

The most efficient way of determining the binding efficiency is by measuring the OD-280 of the antibody dilution before and after coupling.

If you boil the coupled beads in SDS-PAGE buffer for 5 min you will dissociate the coupled antibody, but also streptavidin subunits from the beads. The Streptavidin subunits are around 13 kDa and will also show up on a PAGE gel. Did you not see any protein on your gel? How did you stain the gel? How much sample did you load? Did you include any controls?

Kind Regards

Kristina