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Prepare a total cell lysate as described under Western (Immuno-) blotting procedure. |
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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. |
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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. |
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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). |
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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. |
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Wash pelleted matrix 2 times with 500 µl of PBS, each time repeating the above centrifugation and aspiration steps. |
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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. |
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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. |
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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. |
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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. |
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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. |
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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 |
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After transfer, block/wash membrane with TBST (10x TBST: sc-24953) for 1 hour, changing TBST once half way through the incubation. |
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Dilute WB antibody with ImmunoCruz™ IP/WB Optima E Dilution Buffer (provided), add to membrane and incubate for 1-2 hours at room temperature. |
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After incubation, wash 3x with 1x TBST, 5 minutes per wash. |
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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. |
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Wash membrane 3x with TBST, 5 minutes per wash. |
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Wash membrane once with 1x TBS (10x TBS: sc-24951) for 5 minutes. |
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Incubate membrane in Western Blot Luminol Reagent: sc-2048 according to Luminol data sheet. |
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