Fluorescent Western Blotting Technical Support

NOTE: For the best possible results, Cell Signaling Technology (CST) strongly recommends using our optimized application-specific protocols for each product. These protocols are the result of extensive in-house validation performed at CST and ensure accurate and reproducible results.

Detection of Western blots using fluorescent labeled secondary antibodies can allow for more accurate quantification and two-color multiplexing using primary antibodies from different species. Phosphorylation and total protein levels may be determined on the same membrane, and regulation of different targets at different molecular weights can be examined simultaneously. There can also be significant long-term advantages in terms of digital data storage as well as the reduction of costs for chemiluminescent substrates, x-ray film, and darkroom facilities.

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Many CST antibodies are being tested by Western blot using near-infrared dye-conjugated secondary antibodies and a slightly modified immunoblotting protocol. We have found that omitting Tween-20 during the blocking step is the only significant change necessary. Full details can be found in our Fluorescent Western Immunoblotting Protocol (Primary Ab Incubation In BSA) and our Fluorescent Western Immunoblotting Protocol (Primary Ab Incubation In Milk). We have also found that prestained standards such as our Prestained Protein Marker, Broad Range (Premixed Format) (#7720) are autofluorescent at near-infrared wavelengths.

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. Some antibody pairs may not work together due to overlapping epitopes, interference caused by primary and/or secondary antibodies, or incompatibilities in primary antibody incubation buffers.

For assistance in troubleshooting, please have the information below ready when you contact us. Technical Support can be reached by toll free phone at 877-678-8324 or 877-678-TECH, fax at 978-867-2400 or e-mail at support@cellsignal.com.

General Questions

1. Catalog number.
2. Lot number and reference date printed on tube.
3. How long have you had the product?
4. At what temperature was the product stored?
5. Was the product aliquotted?

Sample Information

1. Cell type used for the sample lysate: (cell line, primary, whole tissue).
2. Sample species.
3. Induction/drug treatment used.
4. Concentration of the drug used.
5. What were the time points of induction?
6. Positive control used.
7. Lysis buffer used: (SDS Sample Buffer, CST Cell Lysis Buffer, CST Chaps Buffer, CST RIPA Buffer, other).
8. What specific phosphatase inhibitors, if any, were included in the lysis buffer?
9. Were the samples sonicated?
10. Amount of protein lysate loaded per well.
11. Date of lysate preparation.
12. Lysate storage temperature: (4°C, -20°C, -80°C, other).
13. Has this antibody previously worked on these samples?

Protocol Information

1. Percentage of acrylamide in the gel.
2. Which membrane was used: (Nitrocellulose, PVDF, Nylon, other).
3. What is the pore size of the membrane being used for transfer?

Blocking

1. Components of the blocking buffer.
2. What was the blocking time?

Primary Antibody Incubation

1. Primary antibody dilution.
2. Components of the primary antibody dilution buffer.
3. Incubation time for the primary antibody.
4. Washing buffer and time after primary antibody incubation.

Secondary Antibody Incubation

1. Secondary antibody source.
2. Secondary antibody dilution.
3. Components of the secondary antibody dilution buffer.
4. Incubation time for the secondary antibody.
5. Washing buffer and time after secondary antibody incubation.

Detection

1. Which detection system was used.
2. Supplier of the detection system.
3. Settings used for image acquisition.
4. What is the appearance of the scanned membrane? Please include a well-labeled image in your email if possible.