Immunoprecipitation Protocol

Catalog Number: FGI-RABIMM10

 

Overview
Product	RabIMM-10™
Description	RabIMM-10™ is a rabbit antibody Immunoprecipitation kit.
Kit Contents
Micro-Spin Columns with Support	10 pieces
Antibody Binding Buffer	40 mL
Antibody Wash Buffer	60 mL
Protein A Regeneration Buffer	10 mL
Immobilized Protein A Slurry	2 mL
Antibody Neutralization Buffer	1 mL
Immobilized Goat anti-Rabbit IgG	1 mL (.5 mL in 50% slurry)
Antigen Elution Buffer	25 mL
Pre-Clearing Buffer	10 mL
SDS-PAGE Sample Buffer	2 mL

  *Antigenic peptide or competing antigenic protein must be provided by investigator

 

Protocol

 

The RabImm-10™ kit involves the following steps:

1. Preparation of Antigen-Antibody complex
2. Separation of Antigen-Antibody complex from the unbound sample proteins
3. Dissociation and elution of Antigen-antibody complex
4. Selective removal of immunoglobulins form immunoprecipitate
5. Regeneration of Proteins A matrix
6. SDS-PAGE and antigen detection

 

Cell Lysate Preparation

1. Solubilize tissue or cells in SolOBuffer™ (Cat # FGI-1958) at 1-2.5mg/ml concentration.
2. Mix 1 µl of Protease Inhibitor A and 1 µl of Protease Inhibitor B to 2 ml of SolOBuffer™ just before tissue or cell lysis. 
3. Polytrol or sonicate to solubilize proteins. Centrifuge at 12,000X for 2 minutes to remove cell debris.

 

Preparation of Antigen-Antibody Complex

 

1. Incubate the primary antibody (0.5-2.0 µg IgG) with the sample lysates (100-250 µl) for at least 1 hour allowing antigen to bind to antibody.  Incubation times vary depending on the target proteins, affinity of antibody to the antigen and ionic strength of incubation medium. 
2. Mix Protein A slurry gently in the bottle using a swirling motion and add 0.4 mL slurry t spin column tube.
3. Place the spin column in the micro-centrifuge tube (provided) and centrifuge at 14,000X for 1 minute.
4. Remove the spin column and discord the solution from the collecting tube. Place the column back in the same tube and wash column with 0.5 mL Antibody Wash Buffer.
5. Close the cap on spin column and mix by gentle end over end movement on a rocker for 1-2 minutes.
6. Spin the column for 1-2 minutes at 14,000X and discard the liquid from tube.
7. Wash column two more times with Antibody Binding Buffer.
8. Place Protein A spin column in a new micro-centrifuge tube.

 

Binding of Immune Complex with Protein A

 

1. Add the prepared immune complex to the Protein A column. Maximum volume applied on the Protein A column should not be more than 300 µl. 
2. Incubate for 30 minutes with shaking.  Centrifuge the spin column in the collection tube at 14000X for 1 minute. Save the flow through fraction collected in the collection tube for future analysis.
3. Wash Protein A with 500 µl of antibody binding buffer by inverting the closed tubes until all the Protein A is in slurry again.  Centrifuge and discard the solution that is collected in the collection tube.
4. Repeat wash step two more times using antibody binding buffer in the same collection tube and place the spin Protein A column in a new collection tube.
5. Before elution make sure that washing is complete by checking the OD280 of the last wash. If the OD280 is higher than the wash buffer, additional washes are necessary to avoid contamination with the Protein A unbound proteins.

 

Elution of the Immunoprecipitated Material

 

1. Add 190 µl of elution buffer to the spin column to elute immunoprecipitated material
2. Mix by gentle turning of the tube end over end for 5 minutes.
3. Spin the tube at 14000X in a new clean collecting tube.
4. Neutralize the elution buffer immediately by adding 20 µl of neutralization buffer and inverting it several times for complete mixing.

 

Removal of Primary Antibody from the Immunoprecipitate

 

1. Pre-clear 100 µl of Goat anti-Rabbit sepharose beads in 1 mL of pre-clearing buffer by incubating for 15 minutes at room temperature. Centrifuge and wash the beads with antibody wash buffer
2. Centrifuge and remove supernatant
3. Wash beads with 200 µl of wash buffer twice
4. Pellet beads by centrifugation at 12,000-14,000X for 5 minutes and discard the supernatant
5. Prepare antigenic peptide solution (10 mg/mL) in appropriate buffer.  Most of the peptides will dissolve in 50 mM Bicarbonate buffer (pH 8.0).  Alternatively, the peptides can be dissolved in a minimum volume of Dimethylfluoride and then diluted to 10 mg/mL concentration in PBS.
6. Add 100 µl of antigenic peptide along with antibody neutralization buffer.
7. Add 50 µl of binding buffer to the same tube and transfer the contents to the tube containing the pre-cleared beads.
8. Incubate end over end for 90 minutes at room temperature or overnight at 4⁰C in a rotary shaker.
9. Centrifuge and remove the supernatant for SDS-PAGE.  If the sample is dilute, concentrate by Freeze drying to a minimal volume and then reconstitute in SDS-PAGE buffer.

 

SDS-PAGE and Western Blotting

 

1. Dissolve the immunoprecipitated material in reducing SDS-Page Sample buffer.  Use 1 part SDS sample Buffer and 1 part sample.
2. Boil the sample for 3 minutes at 100⁰C before electrophoresis

 

Regeneration of Protein A Column

 

1. The Protein A Spin Columns can be regenerated by washing with 500 µl of Antibody Wash Buffer three times. 
2. Centrifuge the column after each wash at 14,000X for 2 minutes.
3. Add 500 µl of regeneration buffer and incubate the column with mixing end over end for 5 minutes.
4. Centrifuge and wash the column again once with 500 µl distilled water and twice with 500 µl Binding Buffer.