Antibody Stripping Buffer

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Overview

  • This unique buffer provides gentle method for removal of primary and secondary antibodies to allow several reprobings on the same membrane.
  • Gentle buffer will not alter the antigen for subsequent immunoprobing.

 

Applications

Western Blot is widely used to detect and compare proteins from a complex mixture utilizing antibody detection on a membrane. Chemiluminescence has become an easy and sensitive method of detection compared to other analysis. Because of the nature of chemiluminescence detection, it is possible to reprobe the separated protein mixture on the membrane. Conventionally, Western blots have been stripped using extremely harsh conditions that may alter the antigen for subsequent immunoprobing. Gerard Biotech's Stripping buffer is a novel formula provides a gentle method of removing primary and secondary antibodies membranes that allows several reprobings on the same membrane

 

Products and Pricing

Product
Size
Catalog

Price
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Antibody Stripping Buffer
30mL solution
ST013
Antibody Stripping Buffer
500mL solution
ST010

 

Protocol

IMPORTANT: Optimization of incubation time is essential for bets results.

IMPORTANT : If the blot cannot be stripped immediately after chemiluminescence detection, the blot can be stored in PBS or TBST at 4oC until the stripping procedure is to be preformed.

1. Place the blot to be stripped in the Stripping Buffer. Add Stripping Buffer, to fully immerse the blot.

2. Incubate the blot in the Striping buffer for 5-15 min at room temperature with strong shaking.

IMPORTANT: In general, higher affinity antibodies or large quantities of detected protein will require longer incubation time for stripping.

3. Empty the Stripping Buffer.

4. To wash, add 0.3 ml of dH2O and shake vigorously.

5. Repeat Steps 4 five more times.

Complete removal of the HRP label monitoring: After Step 5 incubate the membrane with fresh chemiluminescence reagents and expose to film. If no signal is detected with a 5 min exposure, the HRP conjugate has been successfully removed from the antigen or primary antibody

Complete removal of primary label antibody monitoring: After Step 5 incubate the membrane with the HRP-labeled secondary antibody, followed by a wash in wash buffer. Incubate the membrane with fresh chemiluminescence reagents and expose to film. If no signal is detected with a 5 min exposure, the primary antibody has been successfully removed from the antigen.

IMPORTANT: Analysis of the successful removing of immunoprobes is recommended to prevent removal of the antigen or the unsuccessful removal of the antibodies.

6. If signal is detected in the two experiments describe above, place the blot back into Stripping Buffer for additional 5-15 min.

7. After it has been determined that the membrane is free of the immunodetection reagents, a second immunoprobing can take place. Start the second immunoprobing with reblocking of the blot.

IMPORTANT: The blot can be stripped up to 5 times. However, longer exposure times or more sensitive chemiluminescence substrate. Actually, reprobings may result in a decrease in signal if antigen is labile. Analysis of the individual system is required.

 

Performance Results

 

Stripping of Westren blot by Stripper Buffer and ReWestren blot: (A) Whole HeLa extract was loaded on 10% SDS-PAGE as the indicated amount. After gel separation it was blotted on a nitrocellulose membrane by a semi dry blotter. The membrane was blocked by 1% fat-milk for 1 hour, washed for 15 min and two more times for 5 min with TBST. The first immunoblot was preformed with rabbit anti actin antibody for 1 hour (dilution 1: 500) and washed. Goat anti rabbit IgG conjugated to HRP was used as the secondary antibody (dilution 1: 5000) for the chemiluminescence detection. The immunoblot was exposed to film for 15 min. The scanned aoutoradiogram is presented. (B) The first immunoblot was stripped by immerse the blot in the Stripper Buffer for 7 min, and washed for 5 min, 5 times. After stripping, the immunoblot was immersed in chemiluminescence reagent and the blot was exposed to film for 60 min. The scanned aoutoradiogram is presented. (C) Reprobing the first immunoblot (second immunoblot) was blocked again by 1% fat-milk for 1 hour, washed for 15 min and two more times for 5 min with TBST. The second immunoblot was preformed with rabbit anti eIF2 antibody for 1 hour (dilution 1: 500) and washed. Goat anti rabbit IgG conjugated to HRP was used as the secondary antibody (dilution 1: 5000) for the chemiluminescence detection. The immunoblot was exposed to film for 15 min. The scanned aoutoradiogram is presented. (*) Marks are unspecific detections.

NOTES:

1. All incubation was made at room temperature

2. All incubation was made in shaking.

 
     
     
     
     

 

 
 
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