Does anyone have any advices on bromodeoxyuridine beads blocking? I am trying to detect transcripts with bromouridine (BrU) incorporated, but keep getting high unspeficic binding - meaning transcripts without BrU added, only uridine.

I use BrdU (IIB5) antibody from Santa Cruz: sc-32323 AC, this is coupled to agarose beads.

My blocking protocol goes as follows: BrdU beads are blocked for 1h at room temperature on the rotatator, in blocking buffer containing: 0.25× SSPE, 0.05% Tween 20, 37.5 mM NaCl, 1mM EDTA, 0.1% polyvinylpyrrolidone and 0.1% BSA (Ultrapure BSA in solution, stock 50mg/ml). Buffer has the RNase inhibitor added before use.

After beads blocking there are several of washing steps (all buffers have RNase inhibitor added right before use and are stored on ice):

1. Binding buffer 0.25× SSPE, 0.05% Tween 20, 37.5 mM NaCl, 1mM EDTA

2. Low salt wash buffer: 0.2× SSPE, 0.05% Tween 20, 1mM EDTA

3. High salt wash buffer: 0.2× SSPE, 137.5 mM NaCl, 0.05% Tween 20, 1mM EDTA

4. TET buffer: TE (0.01 M Tris, 0.001 M EDTA, pH 7.4) + 0.05% Tween 20.

5. Elution buffer: 50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 20 mM DTT, 1 mM EDTA

I follow the protocol of Gardini, 2017 Chapter Global run-on sequencing (GRO-Seq)

Any advices are highly appreciated!