Western Immunoblotting (Fluorescent) Protocol

For western blots, incubate membrane with diluted primary antibody in either 5% w/v BSA or nonfat dry milk, 1X TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight.

NOTE: Two-color western blots require primary antibodies from different species and appropriate secondary antibodies labeled with different dyes. If the primary antibodies require different primary antibody incubation buffers, test each primary individually in both buffers to determine the optimal one for the dual-labeling experiment.

A. Solutions and Reagents

NOTE: Prepare solutions with purified water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
2. 1X SDS Sample Buffer: (#7722, #7723) 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red.
3. Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5).
4. 10X Tris Buffered Saline with Tween 20 (TBST-10X): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH₂O, mix.
5. Nonfat Dry Milk: (#9999).
6. Blocking Buffer: 1X TBS with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBS and mix well. Tween-20 cannot be present in the blocking-buffer because it can auto-fluoresce and increase non-specific background. After the blocking step, Tween-20 can be re-introduced to subsequent diluent buffers
7. Wash Buffer: 1X TBST.
8. Bovine Serum Albumin (BSA): (#9998).
9. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA or 5% nonfat dry milk as indicated on primary antibody datasheet; for 20 ml, add 1.0 g BSA or nonfat dry milk to 20 ml 1X TBST and mix well.
10. Secondary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
11. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
12. Blotting Membrane: This protocol has been optimized for nitrocellulose membranes (recommended). PVDF membranes may also be used. Pore size 0.2 µm is generally recommended.

B. Protein Blotting

A general protocol for sample preparation.

1. Treat cells by adding fresh media containing regulator for desired time.
2. Aspirate media from cultures; wash cells with cold 1X PBS; aspirate.
3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl per plate of 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
6. Microcentrifuge for 5 min.
7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

NOTE: Loading of prestained molecular weight markers (#7720, 10 µl/lane) is recommended to verify electrotransfer and to determine molecular weights. Prestained markers are autofluorescent at near-infrared wavelengths.

8. Electrotransfer to nitrocellulose membrane.

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm²) of membrane; for different sized membranes, adjust volumes accordingly.

1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with 15 ml of TBS/T.
4. Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
5. Wash three times for 5 min each with 15 ml of TBS/T.
6. Incubate membrane with fluorophore-conjugated secondary antibody (1:5000–1:25,000 dilution of 1 mg/ml stock) in 10 ml of secondary antibody incubation buffer with gentle agitation for 1 hr at room temperature.
7. Wash three times for 5 min each with 15 ml of TBS/T.

D. Detection of Proteins

1. Drain membrane of excess TBS/T and allow to dry.
2. Scan membrane using an appropriate fluorescent scanner following the manufacturer’s recommendations.