Western Blotting and Immunoprecipitation
Western blotting is a widely used technique to detect levels of protein expression in cell or tissue extract. This technique measures protein levels in a biological sample through antibody binding to a specific protein of interest.
Our rigorous validation protocols help us provide specific and reliable antibodies, which helps you get reproducible and meaningful data every time.
Validation: Our antibodies are recommended at optimal dilutions using specific buffers. This data is generated through extensive testing of various experimental parameters.
Controls: We recommend positive and negative control cell extracts so that you can test your antibody performance and optimize experiment conditions.
Protocols: We offer detailed protocols so that you can start your experiments quickly and minimize time and reagent waste.
Companion Products: We offer a comprehensive list of companion products that are validated in-house with our protocols so that you can get the reagents you need to complete your experiment.
Technical Support: Our knowledgeable technical support scientists are a phone call or email away, so you have a partner at the bench.
Akt Western Blotting Example
Western blot of extracts from various cell lines, untreated or treated as indicated with LY294002 #9901 (a PI3 kinase inhibitor), Human Insulin-like Growth Factor I (hIGF-I) #8917 (which activates Akt via IGFIR binding), or Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (which induces the PI3K/Akt pathway through receptor binding), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (left) and Akt (pan) (C67E7) Rabbit mAb #4691 (right).
Immunoprecipitation
Immunoprecipitation (IP) is a technique used to enrich a specific protein from a heterogeneous cell or tissue extract using a target specific antibody. Co-immunoprecipitation (co-IP) is the pull down of intact protein complexes. IP and co-IP are valuable and widely used techniques to identify protein-protein interactions and novel members of protein complexes.
CST supplies high quality antibodies for IP and co-IP experiments that are validated using detailed protocols. The high degree of antibody specificity coupled with the availability of thoroughly validated companion products including IP beads and buffers ensure reliable IP and co-IP data to elucidate protein-protein interactions and protein complex composition. To save time and reagents, several antibodies are available conjugated to sepharose or magnetic beads for direct IP and co-IP experiments.
Custom conjugation of antibodies to sepharose and magnetic beads are available – please contact us for more information.
Immunoprecipitation Data
Figure A. Immunoprecipitation of Neuropilin-1 from PC-3 cell extracts, in the absence of primary antibody (lane 2), using Neuropilin-1 (D62C6) Rabbit mAb #3725 lot 1 (lane 3), or Neuropilin-1 (D62C6) Rabbit mAb #3725 lot 2 (lane 4). Lane 1 is 10% input. Western blot analysis was performed using Neuropilin-1 (D62C6) Rabbit mAb #3725 lot 2.
Figure B. Immunoprecipitation of α-Synuclein from mouse brain tissue extracts, in the absence of primary antibody (lane 2), using α-Synuclein (D37A6) XP® Rabbit mAb #4179 lot 1 (lane 3), or α-Synuclein (D37A6) XP® Rabbit mAb #4179 lot 2 (lane 4). Lane 1 is 10% input. Western blot analysis was performed using α-Synuclein (D37A6) XP® Rabbit mAb #4179 lot 2. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used to avoid recognition of rabbit IgG heavy and light chains by western blot.
Figure C. Immunoprecipitation of caspase-3 from extracts from Jurkat cells treated with Etoposide #2200 (25 µM, 5 hr) using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2), Caspase-3 (8G10) Rabbit mAb #9665 lot 3 (lane 3), or Caspase-3 (8G10) Rabbit mAb #9665 lot 6 (lane 4). Lane 1 is 10% input. Western blot analysis was performed using Caspase-3 (8G10) Rabbit mAb #9665 lot 6. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used to avoid recognition of rabbit IgG heavy and light chains by western blot.
Co-Immunoprecipitation Data
The co-immunoprecipitation data shown above is the pull-down of the mTORC1 complex using antibodies targeting various complex components including: GβL, mTOR, and Raptor. All data was generated using HeLa cell extracts and the antibodies indicated below. Lane 1 is 10% input, lane 2 is the pull-down.
Figure A. IP Pull-down: GβL (86B8) Rabbit mAb #3274. Western Probe: mTOR Antibody #2972
Figure B. IP Pull-down: mTOR Antibody #2972. Western Probe: Raptor (24C12) Rabbit mAb #2280
Figure C. IP Pull-down: Raptor (24C12) Rabbit mAb #2280. Western Probe: GβL (86B8) Rabbit mAb #3274
