Western Blotting Protocol
Introduction
Western blotting, also called protein blotting or immunoblotting, is a method of detecting certain proteins in complex samples based on the specific combination of antigens and antibodies.Owing to the high resolution of SDS-PAGE and the high specificity and sensitivity of solid phase immunoassay, western blotting has become a routine technique for protein analysis. Western blotting is frequently employed for protein identification and enables qualitative and semi-quantitative analysis of the protein. Combined with chemiluminescence detection, the difference of the same protein expression in multiple samples can be compared at the same time.
General rule: Blocking 1h in 5% nonfat milk/PBST, primary antibody overnight in primary antibody dilution buffer, wash in PBST and secondary antibody in 5% nonfat milk/PBST for 1h RT.
Sample preparation.
1. Prepare the IntactProtein™ lysis buffer by adding 2 μL of Reagent A into 1 mL of Reagent B immediately before use. Mix thoroughly by vortexing and place on ice.Tips: Calculate the volume of the lysis buffer you need as per Step 3; discard the unused buffer after use.
2. Discard the cell culture medium and wash the cells twice with ice-cold PBS.
3. Place the culture dish/plate on ice or ice water and add 1 mL of the premixed lysis buffer per 5x10⁶ cells (e.g. add 300 μL of lysis buffer to a 35 mm dish containing 1x10⁶ cells). Keep the dish/plate on ice for an additional 5 min and swirl occasionally to allow the lysis buffer to completely cover the cells.
4. After 5 min of lysis, scrape the cells off the dish/plate using a clean plastic scraper and collect the lysate into a centrifuge tube.
5. Vortex the lysates thoroughly (3 x 10 sec) and place the lysates on ice or ice water for another 10 min to complete the lysis.
6. Heat the lysates on a 95°C heat block for 5 min.
7. Cool the lysates on ice or ice water for 3 min.
8. Centrifuge the lysates at 13, 000g for 5 min at 4°C.
9. Measure the protein concentration using a NanoDrop spectrophotometer or SDS compatible protein assay.
10. Store the lysates at -20°C for future use or use immediately for further analysis.
Membrane Blocking
1. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
2. Wash four times for 10 min each with 25 ml of PBST.
Antibody Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution) in 5 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash four times for 10 min each with 25 ml of PBST.
3. Incubate membrane and secondary antibody (such as HRP-conjugated goat anti-rabbit secondary antibody,Cat#201) in 5 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
Detection of Proteins
1. Wash the membrane bound with antibodies four times for 10 min each with 25 ml of PBST.
2. Prepare ECL Reagent (such as FeQTM ECL Substrate Kit, Cat#226) . Add equal volumes of Reagent A and Reagent B in a small tray. Mix well.
3. Immerse the membrane in the small tray containing the ECL Reagent, ensuring it is fully exposed to the reagent, and then expose to X-ray film or BioRad imaging system.
