Western (Protein) Blotting
Protocols for GE Osmonics Nitrocellulose Membranes
with NitroPure, NitroBind, NitroME, NitroPlus Nitrocellulose Membranes
Protocols are often provided for Western blotting onto nitrocellulose and
nylon membranes, but GE Osmonics recommends that most Western blot procedures
be performed on GE Osmonics NitroBind or NitroPure membranes to reduce the
potential for high backgrounds.
Gel Preparation
Gels may be stained before transfer with Coomassie Blue, or after transfer
with Fast Green, Amido Black, or any other appropriate stain. Soak the gel
for 1 hour in a transfer buffer made of: 25 mM Tris-HCl/pH 8.0, 0.15 M glycine,
20% methanol.
Membrane Preparation Transfer
Completely soak the membrane in deionized water, and then in transfer buffer.
Electroblotting
Assemble the membrane and gel in the electroblotting unit.
Place the membrane on the anode (positive) side of the gel. Transfer according
to manufacturer's instructions. Remove and wash thoroughly with transfer
buffer.
Capillary Blotting
Prepare gel assembly by the method of Southern. Transfer for 2 hours to overnight.
Use transfer buffer of 10 mM Tris-HCl/pH 7.5.
After the transfer step, determine transfer efficiency by staining the blot
or gel by standard methods.
Blocking Procedures
Step 1: First Wash
Block the blot in PBS buffer (0.9% NaCl, 10 mM sodium phosphate/pH 7.2) containing
5% nonfat dry milk for 1 hour, with gentle agitation. Tween-20 may also
be added to enhance blocking.
Step 2: Primary Antibody Binding
Remove the PBS buffer solution from blot completely. Dilute the first antibody
in 50 ml of fresh PBS buffer solution. Incubate the blot in the PBS blocking
buffer/antibody solution for 1 hour at 37°C with gentle agitation. Use
a ratio of 5-10 ml of solution to 100 cm² of membrane.
Step 3: Second Wash
Wash the membrane in 100 ml of fresh PBS buffer solution (without antibody)
with 0.1-0.3% Tween-20. Agitate in a shaker for 5 minutes. Repeat the wash
step 2 times. (Note: Increasing the number of short washes reduces the
potential for high backgrounds).
Detection
Thoroughly remove the PBS buffer solution and overlay the blot with an antispecies
(second) antibody, or with protein A (radiolabelled or enzyme linked) for
1-2 hours at room temperature with gentle agitation.
The final concentration of radiolabelled second antibody solution should
be 1-2 x 105 dpm/ml of PBS buffer solution. Enzyme-linked second
antibody solutions should be made at a 1:1000 titer in PBS buffer solution.
Repeat the wash step described in the procedure above.
Signal Development
The choice of signal development method is dependent on the type of probe
used. Radiolabelled probes are developed and quantitated by autoradiography.
Enzyme-conjugated labels (horseradish peroxidase or alkaline phosphatase)
are developed and quantitated with the appropriate substrate solution. For
further details, please call GE Osmonics (877-Lab-Store) for technical assistance.
Probe Removal
Do not allow the filter to become dry, or irreversible binding of the probe
will result.
Wash the membrane at 60°C for 30 minutes in 0.05 M sodium phosphate/pH 6.5,
10.0 M urea, 0.1 M 2-mercaptoethanol, or wash the membrane in 0.2 M glycine-HCl,
0.5 M NaCl for 5 minutes. Rinse in 0.1 M NaOH or 0.5 M Tris for 10 minutes.
Please call GE Osmonics' Labstore Support at (800) 444-8212, by fax (952) 988-6662 or email us for questions, pricing and availability. For international support and distributor locations, call +1-952-988-6665 or email us.
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