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RENEW STRIPPING BUFFER

Introduction

RENEW STRIPPING BUFFER is a ready-to-use solution allowing primary and secondary antibodies to be quickly removed from nitrocellulose and PVDF membranes.

Features

  • Robust formulation for a fast stripping of most antibodies
  • Non toxic and odorless formulation

Chemiluminescent Western blots can be stripped with reagents that remove affinity-bound primary and secondary antibodies.
RENEW is a stripping buffer which is strong enough to dissociate bound antibodies, but at the same time, minimizes the loss of transferred target proteins on the nitrocellulose or PVDF membrane.
By stripping and reprobing, there is no need to waste precious or expensive samples by running multiple gels, in order to probe for different targets.
A single blot can be stripped with RENEW to remove primary antibodies, thus allowing a reprobing with a new primary antibody or to optimize the antibody concentration after obtaining initially poor results.
Success in stripping requires an accurate analysis of the system (cell type, amount of loaded protein, affinity of the antibodies,…). Optimization of both incubation time and temperature is essential to ensure the complete removal of antibodies while preventing damages to the antigen.
Notice that reprobings could require longer exposure times or a more sensitive ECL substrate, since the signal could be lower than the initial one.

Protocol summary

Wash the blot in 1X TBS or PBS TWEEN®20 for 5 minutes to remove chemiluminescent substrate.
Incubate the blot in RENEW stripping buffer for 20 minutes at RT (optimization of the incubation time and temperature in relation to the affinity antibody-antigen).
Remove blot and wash in 1X TBS or PBS TWEEN®20 for 5 minutes.
Test for removal of antibodies.
Perform a new probing.

Stripping and reprobing

Stripping and reprobing Renew

Stripping and reprobing blots for different protein targets with RENEW STRIPPING BUFFER.
Hela whole cell lysate (abcam®) was diluted to 250 µg/mL in electrophoresis reducing sample buffer and 1:1 serial dilutions were made. 5µL of each dilution (1250 ng to 78 ng of total protein) were separated by SDS-PAGE and the proteins transferred to Amersham™ Protran® Premium 0,45 NC membrane (GE Healthcare). The membrane was blocked with 5% non-fat dry milk in 1X TBS TWEEN®20 Buffer and analyzed by Western blotting using WESTAR ETA C ULTRA chemiluminescent substrate (Cyanagen) with ImageQuantTM LAS 4000 (GE Healthcare). The first target (panel 1) was detected by probing with Rabbit Anti-MAP Kinase (ERK 1/2) antibody (SIGMA) at 1:3000 followed by Anti-Rabbit IgG HRP Linked F(ab′)2 (GE Healthcare) at 1:30000 and imaged. Next, the blot was stripped in RENEW stripping buffer for 20 minutes at RT, washed in 1X TBS TWEEN®20 Buffer, and checked for stripping efficiency (panel 2). The second target (panel 3) was detected by probing with Rabbit Anti-HDAC1 Antibody (abcam®) at 1:2500 followed by Anti-Rabbit IgG HRP Linked F(ab′)2 (GE Healthcare) at 1:30000 and imaged. The blot was stripped again (panel 4) and then probed (panel 5) with Mouse Anti-Vinculin clone Hvin-1 antibody (SIGMA) at 1:2000, followed by Anti-Mouse IgG (whole molecule)–Peroxidase antibody (SIGMA) at 1:20000 and imaged.

Stripping and reprobing - Comparison

Stripping and reprobing Comparison 

Stripping and reprobing performance of RENEW and competitor T.R.
Hela whole cell lysate (abcam®) was diluted to 250 µg/mL in electrophoresis reducing sample buffer and 1:1 serial dilutions were made. Two sets of 5µL/dilution (1,25 µg to 0.78 µg of total protein) were separated by SDS-PAGE and the protein transferred to Amersham™ Protran® Premium 0,45 NC membrane (GE Healthcare). The membrane was blocked with 5% non-fat dry milk in 1X TBS TBS TWEEN® 20 Buffer and analyzed by Western blot using WESTAR ETA C ULTRA chemiluminescent substrate (Cyanagen) with ImageQuant TM LAS 4000 (GE Healthcare). The membrane was probed with Rabbit Anti-MAP Kinase (ERK 1/2) antibody (SIGMA) at 1:3000 followed by Anti-Rabbit IgG HRP Linked F(ab′)2 (GE Healthcare) at 1:30000 and imaged. Following the initial detection, the blot was cut into two strips to separate the serial dilution sets and each part of the blot was stripped, according to manufacturer’s instructions, in either RENEW stripping buffer (20 minutes at RT), T.R. (15 minutes at 37°C). After the stripping procedure, the membrane strips were washed in 1X TBS TWEEN®20 Buffer and checked for stripping efficiency. The membrane strips were reprobed as described above.