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Protocol
Western Blot
Keywords: Lysis, western, blot, transfected cells
Basic protocol for Western Blot.
Required Materials
- SDS-gel , 12%
- iBlot System
- PBS with non-fat milk , 3% w/v
- PBS
- Antibody , primary and secondary
- ECL Plus™
- ChemiDoc-It
Main steps
- (1VGTJDL)
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Run 20-30 µg total protein on in 12% SDS gels (Invitrogen) and transfer…
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Incubate membranes with secondary antibody.
2 hours at room…
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The immunoreactive bands are visualised using ECL plus (Amersham biosciences).…
- (1VGTJDL)
-
Run 20-30 µg total protein on in 12% SDS gels (Invitrogen) and transfer…
Instructions
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Block membranes with 3% w/v non-fat powder milk PBS (PBS/tween).
Wash with PBS, 2X20 min.
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Incubate membranes with primary antibody.
2 hours at room temperature.
Wash 6X3 min in PBS. -
Incubate membranes with secondary antibody.
2 hours at room temperature.
Wash 6x3min with PBS. -
The immunoreactive bands are visualised using ECL plus (Amersham biosciences).
1 to 10 min.
Detection is carried out on a UVP ChemiDoc-It, Imaging system, (UVP Inc). -
Harvest and lyse transfected cells, in lysis buffer.
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Prepare in advance as Triton-X is highly viscous and takes a while to dissolve.
Solution can be kept for 1-2 weeks at +2 to +8°C.
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Run 20-30 µg total protein on in 12% SDS gels (Invitrogen) and transfer to nitrocellulose membranes using iblot system (Invitrogen).
