- Use a cover before and while blocking the plate.
- Vigorous washing is essential; you can effectively remove washing solution remnants by repetitively hitting the plates on a paper towel, wells facing down. This will ensure less cross-reactivity.
- Use a detection system which will not cross react with your sample. If your sample is a lysate from cells that have high levels of alkaline phosphatase, it is better to use an HRP-conjugated antibodies
Tips for Immunoprecipitation & Co-immunoprecipitation
- When performing Co-IP with of membrane, associated proteins then a detergent (NP-40, Triton X-100 etc.) should be maintained along all steps of the procedure.
- Calibrate your lysis buffer ingredients concentration as this can affect the association of antibody to it’s antigen.
- When aiming at detecting novel interacting partners (Co-IP), Pre-clear your sample with prior addition of the washed beads (Protein A/G couples agarose/sepharose beads).
- When working with the beads slurry it is recommended to remove the tip’s edge in order to prevent damaging the beads.
- It is possible to use a gradient of Glycine (up to 1M) to release the antigen from the conjugated antibody in cases the antibody’s interferes with the detection of the antigen or pull down protein.
How BioData can help expedite your work with Antibodies
- Add descriptions and notes in regard to the recommended titer, incubation times, past issues which hampered with assays etc. The free text area in the antibody module’s is the perfect place to add these notes and any other idea you have in regard to the used antibody.
- In BioKM you can link antibodies to a certain experiment. This feature can help determine quickly your needs for the coming experiment. Do you have the antibody in stock? Has someone issued a problem or an improvement when using this protein? You will immediately identify it and adapt while working on your own projects.
- In the antibody’s module of BioKM you can attach files of datasheets, sample images of a successful western blot to help to tackle problems when encountered.
- Assigning specific location to each antibody so fetching a certain antibody is a breeze!
- Fetch specs in regard to the antibody on the fly through the quick view on the right panel
Do you have any tips or tricks that improve antibodies usage? Let us know!
Read Chen's previous post about Antibody Storage and Calibration, how to expedite Western Blots and ELISA washes, and how he uses BioKM to organize his lab's antibody collection. Also read Chen's post with Tips for Working with Western Blots.
Chen Guttman is a Graduate Student at the Zarivach lab in Ben Gurion University. Chen blogs at benchwise and serves as BioData's community liason.