Semi-dry transfer
- prepare following stack
- three whatman filters in cathoden buffer
- gel
- filter (nitrocellulose or PDVF (prior wetting in methanol)
- three whatmen filters in anoden buffer
- transfer the proteins at 60 mA for 1 to 2 h for each mini
gel,
you may control the transfer by the prestained MW marker, or
you may stain the gel with Coomassie
- dissemble the stack
anoden buffer 1:
3.06 g Tris base,
100 ml methanol in 1l
cathoden buffer:
3.02 g Tris base,
5,25 g 6-aminohexansŠure (caprionic acid)
100 ml methanol
Wet transfer
Large proteins (e. g. RhoGEF2) are more efficiently transfered by
wet transfer. If there are problems with transfer efficency, use low percentage
polyacrylamide, add 0.05% or 0.1% SDS to the transfer buffer or reduce
Methanol to 10% in the transfer buffer.
- assemble a stack of whatman filters, gel, membrane, whatman filters
(see above - semi-dry transfer). Make sure that no air bubbles are enclosed. You may
role a pasteur pipette on the stack back and forth.
- enclose into presoaked sponges and put into the cassette of the BIORAD apparatus
filled with transfer buffer.
- transfer the proteins at 200-350 mA (100 V) for 1-2 hours or 100 mA for overnight.
Place the transfer box into
an ice container to absorb the heat. Use a stir bar in the transfer apparatus for
better heat exchange.
Transfer buffer (wet transfer)
25 mM Tris, 190 mM glycine [pH 8.3]
recipe: dissolve 12.12 g Tris base, 57,63 g glycine, 800 ml methanol,
in finally 4000 ml of water..
Antibody binding and detection
- stain the membrane in Ponceau solution for a few minutes
- wash with deionised water (from tap)
- mark the molecular weight markers and orientation.
- blocking: add PBS with 5% (w/v) milk powder,
rock
for 60 min
- add the primary antibody diluted in PBT and 0.5% BSA,
incubate for
2 h at RT or 4¡C overnight
- you may save the solution with the primary antibody,
add
Na-azide
to 0,01%
- rinse 3x with PBT
- wash the membrane 4 x 5 min with PBT
- add the secondary antibody diluted in PBT,
incubate
for 1-2h at RT In case
of background problems, add 0.01% SDS
- rinse 3x with PBT
- wash 4 x 5 min with PBT
- rinse with PBS to remove residual detergent
- wrap in a foil
- scan the blot with the LICOR
Stripping:
In order to develop blots for a
second or third time, primary and
secondary antibodies
are removed with stripping solution.
- blots must stay wet all the time
- incubate for 30 min, 50¡C, in stripping buffer
- block with milk
- add primary antibody
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