Free biotin labeled oligos migrates normally, but samples containing biotin labeled oligos and protein extract stuck in the well.

I would like to investigate the NF-κB binding in whole cell extract of mononuclear cells from spleen. The cells were lysed in RIPA buffer without SDS and lysis was conducted by freezing/thawing and ultrasonic bath sonicator.

EMSA was performed using LightShift® Chemiluminescent EMSA Kit (Cat # 20148, Thermo Scientific). The oligonucleotide sequence containing NF-κB DNA-binding motif. For binding reactions, 20 fmol Biotin-labeled probe was mixed with 1 μg whole cell extract in a total volume of 20 μl containing 10 mM Tris, 5 mM MgCl2, 1 mM DTT, 2,5% glycerol, 0,05% NP-40 and 50 ng Poly[d(I-C)].

The reaction mixtures were applied to a non-denaturing 6% TBE polyacrylamide gel in 0.5xTBE running buffer. Before running the samples, we pre-run the gel for an hour at 100V.

We have tried to reduce the concentration of protein (from 1 µg to 0,25 µg) and doing the electrophoresis on ice.

Many thanks in advance for the help