Far Western analysis of protein interaction experiments
Far Western analysis of protein interaction experiments
When analyzing protein interactions using Far Western blot, the target protein is immobilized on a solid support membrane and then probed with a non-antibody protein. The Far Western blot can be used to identify protein-specific interactions in complex protein complexes. Sourced from Compact Molecular Biology Laboratory Guide (5th Edition)
Operation method
Analyzing protein mixtures
Materials and Instruments
Sample to be analyzed cDNA encoding the target protein (already cloned into an in vitro expression vector) Move Closure buffer I: 0.05% (m/V) Tween-20 dissolved in 1 × PBS buffer, freshly prepared Closure Buffer II: 1 g of bovine serum albumin (BSA; fraction V) dissolved into 100 ml of 1 × PBS, freshly prepared. 1. Prepare the protein sample to be analyzed by melting it into 1 × SDS Sampling Buffer. 2. 2. Separate the samples by SDS polyacrylamide gel electrophoresis. 3. 3. transfer the proteins from the gel to a solid support membrane (e.g., PVDF or nitrocellulose membrane) by semi-dry blotting.) 4. Optional step: after transferring the membrane, stain the membrane for 5 min with 100 ml of freshly diluted 1 × Lysine S. Stain the membrane in a container large enough to hold the membrane and with enough Lysine S to completely pass over the membrane surface. 5. Discoloration: Wash the membrane several times with deionized water until the proteins are clearly visible. Mark important protein bands with a pencil for future reference. Cut off the excess of the membrane. 6. 6. Fade in water for an additional 5 min until the red dye fades. 7. 7. Confine in 200 ml of Closure Buffer I for 2 h at room temperature with gentle shaking. 8. Pour off the solution gently. 8. Gently pour off the solution and add 200 ml of Closure Buffer II. Incubate as in step 7. 9. 9. Pour off Closure Buffer II and rinse gently in 100 ml 1 × PBS. 10. 10. The in vitro translation probe for the target protein radiolabeled with 35S-methionine is prepared according to the manufacturer's procedure below. 11. 11. After translation, the probes are purified by dilution with 500 W Probe Purification Buffer and centrifugation at 10,000 g for 15-30 min at room temperature using a microfiltration centrifuge column. Save a portion of the purified probe for SDS-PAGE electrophoresis (e.g., 2 μl) and scintillation counting (e.g., 2-5 μl). 12. 12. Pre-incubate the membrane with 50 ml of 1 × probe dilution buffer (without probe) for 10 min with gentle shaking at room temperature. 13. 13. Dilute the translated probe with 1 × Probe Dilution Buffer in a volume sufficient to cover the membrane (usually 3 ml). 14. Add the probe to the membrane and incubate for 2 h at room temperature, rotating the tube (if incubating in a tube) or shaking the sealing bag (if incubating in a sealing bag) during the binding reaction. 15. Transfer the membrane to a plastic dish and wash the membrane with 200 ml of 1 × PBS for 5 min at room temperature. 16. 16. Air-dry the membrane and image on X-ray film (radiographic autoradiography) or phosphor screen exposure. Caveat 1. Appropriate safety precautions must be in place when working with radioactive materials. 2. Always wear gloves or use membrane clamping forceps when touching supporting membranes. For more product details, please visit Aladdin Scientific website.
1 × SDS Sampling Buffer Lichtenstein Red S Stain Closed Buffer I Closed Buffer II In vitro Transcription Translation Kit PBS 35S-Methionine Probe Purification Buffer Probe Dilution Buffer
Microfiltration Centrifuge Columns Polyvinylidene Fluoride (PVDF)/Nitrocellulose (NC) Membrane