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General Protocol for Western Blot Kit


TABLE OF CONTENTS

 
Introduction
Contents
Storage
Preparation of Reagents
Principal of Western Blot Kit
Standard Protocol
 


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INTRODUCTION
Phoenix’s Western Blot Kit is designed for the rapid and sensitive immunoenzymatic detection of specific peptides immobilized on nitrocellulose or polyvinylidene fluoride ( PVDF ) membranes, whether transferring from SDS-PAGE / Tricine-PAGE gels after electrophoresis ( western blot ) or bound directly from solution ( dot blot ). The purified primary antibody ( IgG ) used for this assay was raised against a synthetic peptide. A highly purified standard peptide is included with the kit to be used as a positive control or to pre-block the primary antibody. All reagent necessary to produce fast and sensitive results on nitrocellulose or PVDF membrane are included. This kit enable the detection of antigen levels of 500ng or lower.


CONTENTS
  1. Purified rabbit primary antibody ( IgG ), (lyophilized powder), 200 µg
  2. Donkey anti-rabbit IgG whole antibody HRP conjugate, 50 µl
  3. Standard peptide, ( lyophilized powder), 12.8 µg
  4. 10 x TBST pH 7.6 (Catalog No. WB-B001), 100 ml
  5. 10x TBS pH 7.6, (Catalog No. WB-B002), 50 ml
  6. 10x Blocking Buffer and Antibody Diluent, (Catalog No. WB-B003),  50ml
  7. Detection Reagent A, (Catalog No. WB-B004), 125 ml
  8. Detection Reagent B, (Catalog No. WB-B005), 10 ml
  9. Kit Protocol

Notes: This kit supplies sufficient reagents to perform 5 blots.

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STORAGE CONDITIONS:
  1. The primary antibody, Blocking Buffer and Antibody Diluent should be stored below -20° C.
  2. Secondary antibody should be stored at 2 ~ 8 ° C, DO NOT FREEZE!
  3. Detection Reagent A and B are light sensitive. They are stable for 6 months at 2 ~8 ° C in the dark. Keep bottles closed to prevent microbial or dust contamination.

 
PREPARATION OF REAGENTS:
  1. 10 X TBST buffer: Dilute each bottle with 900ml distilled deionized water . Stir to homogeneity.
  2. 10 X TBS buffer: Dilute with 450ml distilled deionized water. Stir to homogeneity.
  3. Primary antibody IgG: Add 200 m l of Antibody Diluent to rehydrate the primary antibody IgG.
  4. Detection Reagent: Mix 10 ml of Detection Reagent A with 5 m l of Detection Reagent B to make enhanced chemiluminescent developing solution. It should be used within 30 minutes.
  5. 10x Blocking Buffer and Antibody diluent: Dilute with 450 ml distilled deionized water. Stir to homogeneity.

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PRINCIPAL OF THE PHOENIX WESTERN BLOT KIT

Phoenix’s Western Blot Kit is based on the enzyme-linked immunodetection of antigen-specific antibody ( supplied with kit ) using anti-IgG secondary antibodies conjugated to horseradish peroxidase ( HRP ), which reacts with a chemiluminescent substrate in the presence of a chemical enhancer. This produces a light signal that can be captured by short exposure to a blue-light sensitive autoradiography film or on the Molecular Imager system. This kit brings a new level of convenience and reliability to a non-isotopic procedure while providing a fast, highly sensitive detection for membrane bound peptides / proteins.

 

 
STANDARD PROTOCOL

Blocking of membrane ( blot )
To saturate nonspecific protein binding sites, incubate the membrane for 30 minutes in Blocking Buffer /Antibody Diluent ( TBST containing 1% blot-qualified BSA ) for nitrocellulose membranes; incubate 60 minutes for PVDF membranes.

Primary Antibody Binding

  1. To bind primary antibody, replace the blocking solution ( which can be re-used several times ) with Blocking Buffer / Antibody Diluent containing appropriate dilution of primary antibody ( recommended primary antibody dilution = 1:500 ~ 1000 ).
    Incubate the blot for 30 ~ 60 minutes with gentle agitation at room temperature (or overnight at 2~ 8 °C).
  2. To remove unbound antibody, wash the membrane three times with TBST for 5 ~ 10 minutes each.

Secondary Antibody Binding

  1. Incubate blot with Blocking Buffer /Antibody Diluent containing the appropriate dilution of anti-rabbit IgG- HRP conjugate for 30 minutes with gentle agitation. We recommend that the secondary antibody dilution be in the range of 1:5000 ~ 10000.
  2. Wash the blot with TBST three times for 10 minutes each to remove unbound secondary antibody.
  3. Briefly rinse twice with TBS to remove Tween 20 from the blot surface.

ECL developing

  1. Mix 10 ml of Detection Reagent A with 5 ul of Detection Reagent B to cover the membranes ( based on 0.125 ~ 0.25 ml / cm2 membrane ). The mixed solution should be used within 30 minutes.
  2. Drain the excess buffer from the washed blot ( membrane ) and place it on a piece of SaranWrap or a glass plate with the protein or peptide side facing up. Add mixed detection solution to the peptide or protein side of the membrane. Make sure the entire surface of the membrane is covered with the mixed detection solution. Incubate for exactly 1 minute without agitation. Drain off excess detection solution and wrap membranes in SaranWrap. Using gloved fingers, smooth out all air pockets on the membrane surface by pressing gently.
  3. Place the blot with the peptide or protein face up, in the film cassette. Work as quickly as possible to minimize the delay between incubating the blot in the mixed detection solution and exposing it to the film. In a dark room, place a sheet of blue light sensitive film on top of the blot, close the cassette and expose for 5 seconds ~ 10 minutes according to signal strength. The second exposure may vary from 1 minute to 30 minutes depending on target signal and background. Perform this step in a dark room using a red safe light.

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