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4: ImmunoCruz™ Optima Immunoprecipitation/Western Blots
Prepare a total cell lysate as described under Western (Immuno-) blotting procedure.
Preclear whole cell lysate (optional): Use appropriate Preclearing Matrix for your kit (sold separately). To approximately 1 ml of whole cell lysate or tissue extract in a 1.5 ml microcentrifuge tube, add 40-50 µl of the suspended (25% v/v) preclearing matrix. Incubate for 30 minutes at 4° C while rotating. NOTE: If the lysate was prepared from cells expressing Igs (i.e., spleen cells or cultured B cells), a preclearing step with Protein A/G agarose should also be performed 2-3 times to ensure complete removal of endogenous Igs.
Pellet IP matrix via microcentrifugation at maximum speed for 30 seconds at 4° C. Without disturbing pellet, transfer desired supernatant (precleared cell lysate) to a new microcentrifuge tube. Store precleared lysate on ice and discard the pellet.
Formation of the IP antibody-IP matrix complex: To a microcentrifuge tube, add 40-50 µl of suspended (25% v/v) IP matrix, 1-5 µg of IP antibody and 500 µl of PBS. Optimal antibody amount should be determined by titration. Incubate at 4° C on a rotator for at least one hour. This step can be performed in parallel with the above preclearing step or performed the day before and allowed to incubate overnight at 4° C.
NOTE: It is necessary that the species of the IP antibody matches the species of the IP matrix included with each ImmunoCruz™ Optima kit. For example, when performing an IP with a mouse antibody, it must be incubated with the Mouse IP Matrix provided (sc-45040 or sc-45042).
After incubation of the IP antibody with the species specific IP matrix, pellet matrix via microcentrifugation at maximum speed for 30 seconds at 4° C. Carefully aspirate and discard supernatant.
Wash pelleted matrix 2 times with 500 µl of PBS, each time repeating the above centrifugation and aspiration steps.
Immunoprecipitation: After the final wash of the IP antibody-IP matrix complex, transfer lysate (100-1000 µg of total cellular protein) to the pelleted matrix and incubate at 4° C on a rotator for one hour to overnight.
After incubation of the matrix and lysate, microcentrifuge at maximum speed for 30 seconds at 4° C to pellet. Aspirate and discard supernatant or alternatively keep supernatant for another IP or testing via western blot.
Wash pelleted matrix 2-4 times with either RIPA Lysis Buffer: sc-24948 (more stringent) or 1x PBS (less stringent), each time repeating the above centrifugation and aspiration steps.
After final wash, aspirate and discard the supernatant and resuspend pellet in 40-50 µl of reducing 2x Electrophoresis Sample Buffer: sc-24945. Boil samples for 2-3 minutes. Note: The immunoprecipitated sample must be completely reduced and denatured for ImmunoCruz™ Optima to work properly.
Perform a quick spin to pellet IP matrix and carefully load supernatant onto gel. Continue with electrophoresis as described under the Western (Immuno) Blotting procedure.
At this stage it is essential that the immunoblotting (primary) antibody matches the species specificity of the HRP conjugated ImmunoCruz™ Optima detection reagent which is unique for each kit. Detect the immunoblotting (primary) antibody using the corresponding HRP conjugated ImmunoCruz™ Optima reagent and Western Blot Luminol Reagent: sc-2048.
NOTE: When using sc-45042 ImmunoCruz™ IP/WB Optima E, the alternate immunoblotting protocol that is specific for this kit must be followed as described below in order to generate desired results.
ImmunoCruz™ IP/WB Optima E Alternate Protocol
After transfer, block/wash membrane with TBST (10x TBST: sc-24953) for 1 hour, changing TBST once half way through the incubation.
Dilute WB antibody with ImmunoCruz™ IP/WB Optima E Dilution Buffer (provided), add to membrane and incubate for 1-2 hours at room temperature.
After incubation, wash 3x with 1x TBST, 5 minutes per wash.
Dilute ImmunoCruz™ IP/WB Optima E Western Blot Reagent (1:1000-1:10000) with ImmunoCruz™ IP/WB Optima E Dilution Buffer (provided), add to membrane and incubate 1-2 hours at room temperature.
Wash membrane 3x with TBST, 5 minutes per wash.
Wash membrane once with 1x TBS (10x TBS: sc-24951) for 5 minutes.
Incubate membrane in Western Blot Luminol Reagent: sc-2048 according to Luminol data sheet.
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SUPPORT PRODUCTS
ImmunoCruz™ Optima Immunoprecipitation/Western Blots
Cruz Marker™ Compatible Secondary Antibodies for Western Blotting

Cruz Marker™ Molecular Weight Standards

Western Blotting Chemiluminescence Luminol Reagent

Prestained Molecular Weight Standards

Western Blotting Membranes

Blocking Reagents for Western Blotting Applications

Gel Incubation Trays

Cruz Blot™ Systems for Western Blotting

Conventional Secondary Antibodies for Western Blotting

Isotype-Specific Secondary Antibodies for Western Blotting

Adult Tissue Extracts for Western Blotting

Immunoprecipitation Reagents

Whole Cell Lysates for Immunoprecipitation

Control IgGs and IgG Conjugates

Purification Reagents

GST-Agarose Preclearing Reagent

Nuclear Extracts for Gel Shift and Western Blotting

PhosphoCruz™ Protein Purification System

Phospho-Enriched Whole Cell Lysates for Western Blotting

Phospho-Enriched Tissue Extracts for Western Blotting

Tissue Extracts for Immunoprecipitation

Acetylation Specific Antibodies

Support Products