Receptor binding assay-hassels with background

Sarah-Jane McKay s.mckay at sct.gu.edu.au
Sun Mar 7 19:19:28 EST 1999


Hi,

I'm attempting to perform whole cell membrane receptor binding assays
using a radiolabelled protein as the ligand.  We are trying to determine
whether a outer cell membrane receptor exists for this protein.  The
protein is 12kDa and is labelled with 35S-meth/cys.  I'm growing cells
in a 96-well format and harvesting using a Tomtec cell harvester to
transfer cells onto a Glass Fiber filter mat - B thickness.  The cells
are grown in RPMI 1640, radiolabelled protein is added and incubated for
a given time before cells are directly harvested.

The trouble I'm having is that the background caused by the
radiolabelled protein alone is very high (40 000cpm) and compared to
when combined with cells (about 50 000 to 60 000cpm).  I suspect that
the protein is too big for this type of mat and that it gets trapped too
easily.

To date I have tried washing with both water and 1X PBS with no
success.  I have also attempted to block the filter mat prior to harvest
using PEI (as this is what was being used in the lab I've been working
in) - the mat is dry when harvesting occurs, and also using blotto (a
general blocking agent comprising of Tris, NaCl and skim milk powder)-
soaked in blotto immediately prior to harvesting.  All to no avail.
Today I will attempt blocking with BSA.

Any other suggestions as to a better method or any alternatives would be
greatly appreciated.

S.McKay




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