BMBMethod:western
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Western Blotting w/Tween 20 Blocking
Tween-20 can be used, in lieu of proteins like BSA or milk, to block both nitrocellulose and PVDF membranes for western blotting
Starting after transfer
Optional: Dry blot to potentially help ‘fix’ proteins
Note: PVDF will likely need re-wetting with 100% methanol if you do this
- Blocking: TBST w/0.5% Tween 20 for 30min
- (Supplement TBST with 1/100th vol of 25% Tween 20 to get to 0.5%)
- This website says you can block in only a few min if you use 2% Tween 20
- 1° = Dilute in TBST, incubate 30-60min at RT
- Wash 3x 5min w/TBST or 1x 10min
- 2° = Diute in TBST, incubate 30-60min at RT
- Wash 3x 5min w/TBST
- Develop w/whatever
Source for Tween concentrations: http://www.agrisera.com/en/info/western-blot.html
You may get much higher signal from this, which would require a more dilute primary and/or secondary antibody.
TBST
| 10mM Tris; pH 8 | 1.21 g/L |
| 150mM NaCl | 8.77 g/L |
| 0.25% Tween 20 | 2.5 g/L |
References
- Batteiger, B., Newhall, W. J., & Jones, R. B. (1982). The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. Journal of Immunological Methods, 55(3), 297–307. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/6820029
- http://www.agrisera.com/en/info/western-blot.html
