Specific applications tips for western blotting & dot blotting
- Do not overwash your membrane as it can eventually strip the antibody from the antigen
- Remember to always add positive control per gel used for easier troubleshooting when there is no detectable bands.
- Calibrate your positive control - certain proteins/tags can have strong signal which can mask your sample’s signals. Load the minimal amount of control which can be detected.
- If reusing antibody solution mark each usage on the tube with a marker; every five usages discard of the solution and prepare new. Also prior to each usage visually check that you don’t have any lumps of “cheese”.
- Use PVDF membrane for analytical purposes (amino terminal sequencing or amino acid content analysis) and for low abundant protein samples.
- It is possible to re-use Ponceau-S stain for the evaluation of the transfer efficacy. However, it should be discarded after 5-10 uses in order to avoid blocking of epitopes on the antigen.
- Want to have both coomassie and antibody staining of the same gel? No problem! Save time by staining your gel with coomassie and after extensive coomassie destaining blot your gel according to common protocol. *Note: This specific tip is compatible with Licor Imager for densitometry measurement. if you are using the licor imager to document the signal use nitrocelulose membranes and DO NOT stain them. (Thanks to Maria Rendon for this tip via Twitter)
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.