Western Blot
Samples:
Gel 1: %, mm SDS PAGE gel – loaded µ1 unless otherwise indicated
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Ran at mA for (See BioRad guidelines for suggested amperages)
Ran at mA for
Gel 2: %, mm SDS PAGE gel – loaded µ1 unless otherwise indicated
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Ran at mA for
Ran at mA for
1. Soaked gels in 1x transfer buffer with MeOH for at least 30min. (0-20% MeOH; 20% standard in our lab)
2. Soaked PVDF membranes in MeOH for ~2min and then in 1x transfer buffer with MeOH.
3. Soaked pads and filter paper in 1x transfer buffer with MeOH.
4. Made gel sandwiches as below and transferred at V for in 1x transfer buffer with MeOH. (Perform at 4C). (Typically 30V overnight)
5. Rinsed membranes briefly in TBS.
6. Blocked in 1x TTBS with 5% (w/v) non-fat dried milk powder and incubated at
on (Typically 2hr at RT or overnight at 4C).
7. Diluted primary Ab in and incubated with membranes at
on . [Dilute in 1x TTBS with either 5% milk powder or 5% BSA depending on manufacturer instruction. Dilute 1:200-1:10,000 (starting dilution typically 1:1000) Incubate on rotary for tubes. Antibody volume 10ml for large tubes. Incubation typically overnight at 4C except for very strong antibodies]
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8. Washed 4x 10min in 1x TTBS.
9. Diluted secondary Ab in and incubated with membranes for
at on . [See above. Dilute 1:1000-1:20,000 (starting dilution typically 1:2000). Typically incubate 1hr a RT]
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10. Washed 4x 10 min in 1x TTBS
11. Developed using the ECL Plus Western Blot Detection System (Amersham Bioscience).
(We also use a Pico detection system; see manufacture instructions)
a. Mixed ml Solution A with µl Solution B. (Need ~3ml for large membrane)
b. Touched corner of membranes to paper towel to remove excess wash buffer
c. Placed membranes on plastic wrap protein side up and flooded them with detection solution (start at one corner and tilt membrane).
d. Incubated at RT for min.
e. Removed excess liquid by touching corner of membrane to paper towel.
f. Placed membranes in page protector.
g. Exposed to film for (1sec up to 1hr; can also use ChemiDoc for 30sec-1hr but detection is slightly less sensitive.)
Current as of 12-15-14