I am trying to validate a Biotin-streptavidin pull down protocol. My downstream application is Western blotting. I am working with HEK-293t cells and BASU. I incubate with streptavidin magnetic beads (overnight)--> wash the beads-->elute--> run on Western.
Unfortunately after applying IRDye streptavidin to label the captured proteins I get an empty gel at the wells of the "Eluted" proteins. I am sure that biotinylation worked since I get a labeling of the input (before streptavidin beads incubation) and the supernatant (after incubation with the beads).
At this point we believe either the Magnetic beads do not bind to the biotinylated proteins or due to a strong affinity they do not discharge the biotinylated proteins. Since the later is more probable we tried the following till now:
- several lysis buffers for protein extraction, from SDS conc. of 0.1-0.4%
- I tried eluting by incubation with 25mM biotin diluted in sample buffer SDS x5, heated at 95 C for 5 minutes, or for 40 minutes
- eluting in 25mM biotin diluted in Tris-Hcl
- eluting in smaple buffer SDS x5 alone
Till now nothing of the mentioned changes had a difference.
Have anyone worked with these NEB-Streptavidin magnetic beads (S1420) and succeeded?
To address your concerns about your protein not binding the beads, have you run the “depleted“ fraction on a western blot? (Add beads, incubate, after removing the beads take an aliquot and run that) you should see loss of protein/biotin if it’s binding the beads, compared to your input.
we’ve used streptavidin dynabeads with success simply using loading buffer and boiling for 10mins, followed by removal of beads by centrifugation.
Murtaza S Nagree Yes we have! But we did not see any difference between the input (a sample of the extracted protein) and the "depleted" (unbound). We think its is due to a low biotinylated proteins from the beginning making it hard to simply see a difference on western, rather than a complete lack of binding between streptavidin beads and biotin.
were you able to see difference between them when you did you experiment?
Unfortunately we did not look for depletion and were directly measuring the protein, not biotinylaton. In other pulldowns, though (His, GST) we have seen depletion.
it doesn’t make sense to me that, due to lower biotinylation efficiency, you would see less depletion if you were measuring biotinylated protein, say using streptavidin-HRP or anti-biotin antibody. Have you tried that?
Murtaza S Nagree I incubate the membrane with IRDye (infrared dye-labled) streptavidin and weren't able to distinguish a difference between the input and the "depleted". so, kinda applying an anti-biotin antibody
Do you have a lane that is a good negative control, for example the same protein/transfection/lysate, but not biotinylated, and does that turn out negative?
If yes, seems like your biotinylated protein just isn’t binding your beads.
If it looks the same as your input, I would argue that you don’t have biotinylation.
Yeah and it does turn out negative.
as I mentioned we thought about it, but believed that its more reasonable that the beads are not releasing the proteins than not binding at all. If from your experience with other beads there was a difference between the input and the depleted then it sounds like my problem with the beads could be more with them binding..
I appreciate your help
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