Hi Sulgi,

I have used ice-cold PBS-CM (PBS containing 0.5mM CaCl2, 2m M MgCl2) instead of just PBS. The calcium and magnesium is to keep the cells more "happy", I have learned that in buffer without a certain amount of calcium and magnesium the cells are much more stressed. Then important if you want to isolate cell-surface proteins, please use the sulfo-form of NHS-LC-biotin

https://www.thermofisher.com/order/catalog/product/21335

NHS-LC-biotin is membrane-permeable (at 4°C not so good, but enough to maybe impair your results). Then oif you results getting worser, maybe "your" NHS is degraded. NHS is quite sensitve against hydrolysis, the powder tend over time to degrade in the lab. Best is to use a pre-aliquoted biotinylation reagent like #21327.

Best regards,

Carsten

Thank you Carsten,

Indeed, I use Sulfo-NHS-LC biotin, no weigh format, so they should be fresh every time I perform the experiment.

Do you incubate biotinlyated protein with Strep-magnetic beads at room temp or in 4°C?

Thank you.

Hi Sulgi,

I incubated my samples with the beads at room temperature. I have read your protocol and can't find anything which should cause your problems, sorry.

Best regards,

Carsten

Thank you for your help Carsten.