What is your protocol for treating western blot membranes with CIP?

CIP-treated membranes are prepared with the following protocol:

REAGENTS:

Quick CIP - #M0525 (New England Biolabs)
rCutSmart™ Buffer - #B6004 (New England Biolabs)
Tris Buffered Saline with Tween® 20 (TBST-10X) #9997

PROCEDURE:

1) Solution Preparation. 1X TBST Wash Buffer: Prepare 1L of 1X TBST by adding 100ml of 10X TBST to 900ml of RODI water and mix well.

2) A duplicate set of lysates are run on SDS-PAGE and transferred to nitrocellulose membrane.

3) After the transfer of proteins to the nitrocellulose membranes and BEFORE THE BLOCKING STEP, prepare 2 incubation vessels:

a) Control/No CIP
b) Treated/+CIP

4) To the control/No CIP vessel, add 900μl of RODI water and 100μl of the 10X rCutSmart™ Buffer that is supplied with the CIP for 1mL total.  If more volume is required, these volumes can be scaled.

5) To the treated/+CIP tube, add 850μl RODI water, 100μl 10X rCutSmart™ Buffer, and 50μl CIP for 1mL total. If more volume is required, these volumes can be scaled.

6) Incubate one membrane in the control/No CIP solution and the other in the treated/+CIP solution for 2 hours at 37C, overnight at 4C, or overnight at 37C.

7) After the CIP treatment, wash the membranes 3 times with 1X TBST and continue with the western blot protocol.  
 

Last updated: March 14, 2024