Western Blot Protocol

Product Manager: Harrison Michael

Protein blotting (WB) is widely used to analyze the expression of specific proteins in cell or tissue extracts. Western blot experiments are time-consuming, and their success has a significant impact on research progress. To this end, we have carefully developed antibodies, provided optimized experimental procedures, reference information, and technical support to ensure the success of your Western blot experiments.

Note: Please refer to the primary antibody product webpage for recommended primary antibody dilution buffers and dilution ratios.

A. Solutions and Reagents

Note: Prepare solutions using reverse osmosis deionized water or water of equivalent grade.

1. 20× Phosphate Buffered Saline (PBS): (P492453) To prepare 1 L of 1× PBS: Add 50 mL of 20× PBS to 950 mL of dH₂O, then mix well.

2. 10× Tris-Buffered Saline (TBS): (T397928) To prepare 1 L of 1× TBS: Add 100 mL of 10× TBS to 900 mL of dH₂O, then mix well.

3. 1× SDS Sample Buffer: SDS-PAGE loading buffer (S750311). Mix the SDS-PAGE loading buffer (reducing, 6×) with the protein sample at a ratio of 1:5. Heat the protein sample in a boiling water bath for 3–5 minutes. After the protein sample is fully denatured, cool it to room temperature and centrifuge at <3000 rpm for 30 seconds. Take an appropriate amount of the supernatant.

4. 10× Tris-Glycine SDS Electrophoresis Buffer: (S301872) To prepare 1 L of 1× electrophoresis buffer: Add 100 mL of 10× electrophoresis buffer to 900 mL of dH₂O, then mix well.

5. 10× Tris-Glycine Transfer Buffer: (T492968) To prepare 1 L of 1× transfer buffer: Add 100 mL of 10× transfer buffer to 200 mL of methanol + 700 mL of dH₂O, then mix well.

6. 1× Tris Buffered Saline with Tween 20 (TBST): Contains 20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6±0.1 (25℃).

7. Tween 20 (T434505)

8. Skimmed Milk Powder (N752146)

9. RIPA Lysis Buffer (R493085)

10. Blocking Buffer: 1× TBST containing 5% w/v skimmed milk powder; to prepare 150 mL, add 7.5 g of skimmed milk powder to 150 mL of 1× TBST and mix well.

11. Washing Buffer: 1× TBST.

12. Bovine Serum Albumin (BSA): (B265993)

13. Phosphatase Inhibitor Cocktail (P777310)

14. Protease Inhibitor Cocktail (P301902)

15. 30% Acrylamide-Bisacrylamide Solution (A665714)

16. Ammonium Persulfate (A112450)

17. PAGE Gel Rapid Preparation Kit (recommended for convenience) (P777326)

18. UltraBio™ SDS-PAGE Precast Gel (Tris-Gly) (recommended for convenience) (E753596)

19. UltraBio™ SDS-PAGE Precast Gel (Bis-Tris) (recommended for convenience, note: buffer is MOPS-SDS Running Buffer T767955) (U776776)

20. Primary Antibody Dilution Buffer: 1× TBST containing 5% BSA or 5% skimmed milk powder (as shown on the primary antibody product webpage); to prepare 20 mL, add 1.0 g of BSA or skimmed milk powder to 20 mL of 1× TBST and mix well.

21. Blue Prestained Protein Molecular Weight Marker (10–250 kDa): (rp210256)

22. Blotting Membranes and Paper: PVDF membranes. Typically, 0.22 µm (P766356) or 0.45 µm (P766361) pore sizes are recommended.

23. HRP-Conjugated Secondary Antibodies: Anti-rabbit (Ab176443); Anti-mouse (Ab179001).

24. Detection Reagents: ECL Plus Ultra-Sensitive Luminescent Solution (E266188 picogram level, W266209 femtogram level).

B. Protein Blotting

General Procedure for Sample Preparation:

(1) Method for Preparing Total Protein from Suspension Cell Lysis

1. Remove the culture dish from the CO₂ incubator and observe the cells under a microscope to ensure the cell confluency >70% and good cell status.

2. Prepare the cell lysis working solution at the required volume on ice; for example, to prepare 50 mL of lysis working solution, add 0.5 mL of protease inhibitor cocktail (P301902) to 50 mL of RIPA Lysis Buffer, add appropriate phosphatase inhibitors as needed, mix well, and use on the same day.

3. Collect the cells in the culture flask into a centrifuge tube, centrifuge at 1200 rpm for 5 min to collect cells.

4. Add 40 mL of pre-cooled DPBS to the centrifuge tube, wash the cells, centrifuge, and discard the supernatant; wash twice.

5. Mix an appropriate volume of lysis solution with the collected cells, gently resuspend the cells, and lyse on a 4℃ rotating incubator for 30 min. 

Note: Generally, 2 to 3 mL of lysis working solution is required for one T75 flask.

6. Repeat until all cells in the culture dishes are collected, and lyse on a 4℃ rotating incubator for 30 min to ensure complete lysis.

7. After lysis, centrifuge at 8000 rpm for 20 min at 4℃.

8. Discard the precipitate and collect the supernatant. Filter the collected lysate through a 0.45 µm filter into a clean centrifuge tube, place on ice, and determine the protein concentration using the BCA method.

9. Concentration and dilution of cell lysate (this step is for lysate with excessively high or low concentration):

o If the concentration >2.5 mg/mL, dilute it to 1.5–2.5 mg/mL with RIPA Lysis Buffer.

o If the protein concentration <1 mg/mL, proceed to steps 10–14.

10. Before using the ultrafiltration centrifuge tube, pipette 3 mL of PBS and gently pipette the tube wall and membrane; balance and place in the centrifuge, set the speed to 5000G, centrifuge for 5 min.

11. Remove the ultrafiltration centrifuge tube from the centrifuge, pour out the filtered and unfiltered PBS, add the lysate solution, balance, and place the ultrafiltration centrifuge tube in the centrifuge so that the membrane faces the center of the rotor.

12. Set the speed to 5000G, centrifuge for 20 min when the ultrafiltration volume is 15 mL (if the original concentration of the lysate is extremely low or high, adjust the centrifugation time as appropriate).

13. After concentration, pipette the concentrate while gently washing the membrane, and transfer to a clean centrifuge tube.

14. Re-determine the concentration of the concentrated cell lysate to ensure it is between 1–2.5 mg/mL. Aliquot as needed and label properly.

(2) Method for Preparing Total Protein from Adherent Cell Lysis

1. Remove the culture dish from the CO₂ incubator and observe the cells under a microscope to ensure the cell confluency >70% and good cell status.

2. Prepare the cell lysis working solution at the required volume on ice; for example, to prepare 50 mL of lysis working solution: add 0.5 mL of protease inhibitor cocktail (P301902) to 50 mL of RIPA Lysis Buffer, add appropriate phosphatase inhibitors as needed, mix well, and use on the same day.

3. Place the culture dish on ice and aspirate the medium from the culture dish with a 10 mL pipette.

4. Add 5 mL of pre-cooled DPBS to the culture dish, wash the cells, and aspirate; wash each dish twice.

5. Add 0.5–1 mL of cell lysis working solution to each culture dish, use a cell scraper to completely scrape the lysed cells from the bottom of the culture dish, and collect them into a clean centrifuge tube (keep the centrifuge tube on ice throughout).

6. Repeat until all cells in the culture dishes are collected, and lyse on a 4℃ rotating incubator for 30 min to ensure complete lysis.

7. After lysis, centrifuge at 8000 rpm for 20 min at 4℃.

8. Discard the precipitate and collect the supernatant. Filter the collected lysate through a 0.45 µm filter into a clean centrifuge tube, place on ice, and determine the protein concentration using the BCA method.

9. Concentration and dilution of cell lysate (this step is for lysate with excessively high or low concentration):

o If the concentration >2.5 mg/mL, dilute it to 1.5–2.5 mg/mL with RIPA Lysis Buffer.

o If the protein concentration <1 mg/mL, proceed to steps 10–13.

10. Before using the ultrafiltration centrifuge tube, pipette 3 mL of PBS and gently pipette the tube wall and membrane; balance and place in the 5804R centrifuge, set the speed to 5000G, centrifuge for 5 min.

11. Remove the ultrafiltration centrifuge tube from the centrifuge, pour out the filtered and unfiltered PBS, add the lysate solution, balance, and place the ultrafiltration centrifuge tube in the centrifuge so that the membrane faces the center of the rotor.

12. Set the speed to 5000G, centrifuge for 20 min when the ultrafiltration volume is 15 mL (if the original concentration of the lysate is extremely low or high, adjust the centrifugation time as appropriate).

13. After concentration, pipette the concentrate while gently washing the membrane, and transfer to a clean centrifuge tube.

14. Re-determine the concentration of the concentrated cell lysate to ensure it is between 1–2.5 mg/mL. Aliquot as needed and label properly.

C．Electrophoresis

2. Preparation of separation gel (taking 10% as an example)

Operation steps: Mix the first four components and vortex gently, add TEMED and immediately pour the gel to 80% of the glass plate height, cover the surface with isopropanol or water to isolate oxygen, and stand at room temperature for 30 minutes.

3.Preparation of stacking gel (5%)

Operation steps: Pour off the liquid on the separation gel, dry the residue with filter paper, prepare the stacking gel and fill the remaining space, insert a comb to avoid air bubbles, and stand for 20 minutes.

Aladdin PAGE Gel Rapid Preparation Kit or UltraBio™ PAGE precast gel can also be selected for greater convenience!

4. Electrophoresis conditions:

· Stacking gel: 80V constant voltage (about 30 minutes)

· Separation gel: 120V constant voltage (about 1–1.5 hours)

5. Membrane transfer (wet transfer example)

· Activate the PVDF membrane with methanol for 30 seconds.

· "Sandwich" structure of gel-membrane-filter paper (avoid air bubbles).

· Transfer at 100V constant voltage for 60 minutes at 4℃ (extend to 90 minutes if the target protein >50 kDa).

· Verification method: Ponceau S staining to check transfer efficiency, observation of prestained Marker bands.

D. Membrane Blocking and Antibody Incubation

Note: Volumes are suitable for a 10 cm × 10 cm (100 cm²) membrane; for membranes of different sizes, adjust the volumes accordingly.

I. Membrane Blocking

1. (Optional) After transfer, wash the PVDF membrane with 25 mL of TBS at room temperature for 5 minutes.

2. Incubate the membrane in 25 mL of blocking buffer at room temperature for 1 hour.

3. Wash three times with 15 mL of TBST, 5 minutes each time.

II. Primary Antibody Incubation

Proceed with the specific steps of one of the following according to the primary antibody used.

(1) For unconjugated primary antibodies

1. Place the membrane and primary antibody (at the appropriate dilution ratio and diluent as recommended on the product official website) in 10 mL of primary antibody dilution buffer, incubate at 4℃ overnight or at room temperature for 1 hour, and shake on a shaker.

2. Wash three times with 15 mL of TBST, 5 minutes each time.

3. Select the appropriate HRP-conjugated secondary antibody (Ab176443 or Ab179001) according to the primary antibody source, incubate in 10 mL of blocking buffer for 1 hour, and shake on a shaker.

4. Wash three times with 15 mL of TBST, 5 minutes each time.

5. Proceed to detection (Section E).

(2) For HRP-conjugated primary antibodies

1. Place the membrane and primary antibody (at the appropriate dilution ratio as recommended in the product data sheet) in 10 mL of primary antibody dilution buffer, incubate at 4℃ overnight or at room temperature for 1 hour, and shake on a shaker.

2. Wash three times with 15 mL of TBST, 5 minutes each time.

3. If detecting biotinylated antibodies or proteins, use Streptavidin-HRP to incubate in 10 mL of blocking buffer at room temperature for 1 hour and shake on a shaker.

4. Wash three times with 15 mL of TBST, 5 minutes each time.

5. Proceed to detection (Section E).

(3) For biotinylated primary antibodies

1. Place the membrane and primary antibody (at the appropriate dilution ratio as recommended in the product data sheet) in 10 mL of primary antibody dilution buffer, incubate at 4℃ overnight, and shake on a shaker.

2. Wash three times with 15 mL of TBST, 5 minutes each time.

3. Incubate the membrane with Streptavidin-HRP (np156148 at an appropriate dilution ratio) in 10 mL of blocking buffer at room temperature for 1 hour and shake on a shaker.

4. Wash three times with 15 mL of TBST, 5 minutes each time.

5. Proceed to detection (Section E).

E. Protein Detection, Taking ECL Western Blot Kit as an Example

1. After completing the secondary antibody incubation, wash the blot membrane thoroughly.

2. Prepare the chemiluminescent detection substrate working solution by mixing ECL-A and ECL-B at a 1:1 ratio by volume as needed (approximately 2 mL of working solution is used for a 10 cm × 10 cm membrane).

3. Discard the washing buffer, add the luminescent substrate working solution to the blot membrane, ensure the working solution covers the entire membrane, and incubate at room temperature for 3–5 minutes.

4. Perform X-ray film exposure in a darkroom, or place the membrane in a chemiluminescent imaging system and perform detection according to the instrument instructions.

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