BMBMethod:western: Difference between revisions
(New page: {{BMBMethodMenu}} ==Western Blotting w/Tween 20 Blocking== ''Starting after transfer''<br> Optional: Dry blot to potentially help ‘fix’ proteins<br> Note: PVDF will likely need re-wett...) |
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==Western Blotting w/Tween 20 Blocking== | ==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<br> | |||
''Starting after transfer''<br> | ''Starting after transfer''<br> | ||
Optional: Dry blot to potentially help ‘fix’ proteins<br> | Optional: Dry blot to potentially help ‘fix’ proteins<br> | ||
| Line 6: | Line 7: | ||
* Blocking: TBST w/0.5% Tween for 30min | * Blocking: TBST w/0.5% Tween 20 for 30min | ||
** (Supplement TBST with 1/100th vol of 25% Tween 20) | ** (Supplement TBST with 1/100th vol of 25% Tween 20 to get to 0.5%) | ||
** [http://www.dako.com/immunovisualization_procedure.pdf 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 | * 1° = Dilute in TBST, incubate 30-60min at RT | ||
* Wash 3x 5min w/TBST or 1x 10min | * Wash 3x 5min w/TBST or 1x 10min | ||
| Line 16: | Line 18: | ||
Source for Tween concentrations: http://www.agrisera.com/en/info/western-blot.html | Source for Tween concentrations: http://www.agrisera.com/en/info/western-blot.html | ||
<br> | <br> | ||
You may get ''much higher'' signal from this, which would require a more dilute primary or secondary antibody. | You may get ''much higher'' signal from this, which would require a more dilute primary and/or secondary antibody. | ||
<br> | <br><br> | ||
'''TBST''' | '''TBST''' | ||
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|0.25% Tween 20||2.5 g/L | |0.25% Tween 20||2.5 g/L | ||
|} | |} | ||
<br><br><br><br><br><br> | |||
==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 | |||
Latest revision as of 05:46, 30 October 2014
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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
