Western blot analysis is a widely used biochemical technique. Its core principle involves transferring protein bands separated by SDS-PAGE onto a solid support membrane for subsequent detection. The procedure generally consists of three main steps: gel electrophoresis, protein transfer (blotting), and immunodetection.

Western blot technology continues to evolve, with significant advances achieved in both transfer methods and immunodetection. Innovations such as double-transfer techniques, tissue section blotting, native electrophoresis, grid-based immunoblotting, and multiplex antigen detection have greatly expanded the applications and development of protein blotting methods

Experimental Steps

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Sample Denaturation and Electrophoresis

1. Prepare the following samples:
   （1）15 μL sample before IPTG induction (without DTT)
   （2）15 μL sample before IPTG induction (with DTT)
   （3）15 μL sample after IPTG induction (without DTT)
   （4）15 μL sample after IPTG induction (with DTT)
2. Add 5 μL of protein loading buffer to each EP tube.
3. Gently mix the samples.
4. Denature the samples in a 100 °C water bath for 3–5 minutes.
5. Vortex briefly for a few seconds and centrifuge shortly.
6. Add 300 mL of running buffer into the electrophoresis tank.
7. Load 10 μL of each denatured protein sample into the wells (left side), and load 5 μL of protein marker.
8. Connect the power supply (red to red, black to black).
9. Run electrophoresis at 80 V initially.
10. When the bromophenol blue dye reaches the separating gel, increase the voltage to 120 V.
11. Stop electrophoresis when the dye front reaches the bottom edge of the gel.

Sample Blotting (Transfer):

1. Cut off the stacking gel and excess gel regions far from the target bands using a scalpel.
2. Soak the gel in pre-cooled transfer buffer (4 °C) for 20 minutes.
3. Cut a nitrocellulose (NC) membrane to the same size as the gel.
4. Soak the membrane separately in pre-cooled transfer buffer (4 °C) for 20–30 minutes.
5. Remove the protective cover and stainless-steel cathode assembly from the transfer apparatus.
6. Assemble the transfer “sandwich” on the anode (platinum side).
7. Carefully place the cathode assembly, reinstall the protective cover, and connect the power supply.
8. Ensure the transfer direction is from cathode to anode.
9. Set the transfer current according to gel size (typically 2–3 times the gel area).
10. Transfer for 45–60 minutes.
11. After transfer, retain the useful portion of the membrane.
12. Cut a small corner on one side of the membrane to mark its orientation.
13. Stain the gel with Coomassie Brilliant Blue for about 50 minutes.
14. Cover the gel with destaining solution and destain for approximately two days to verify whether protein transfer is complete.

Immunodetection:

1. Add 30 mL of TBS-T buffer and 1.5 g of skim milk powder.
2. Place the membrane (front side facing up) into a culture dish.
3. Incubate with gentle horizontal shaking at room temperature for 1 hour.
4. Wash the membrane with TBS-T buffer 3 times, 5 minutes each.
5. Dilute the primary antibody with antibody dilution buffer.
6. Incubate the membrane with the primary antibody at 4 °C overnight with gentle shaking.
7. Wash the membrane with TBS-T buffer 5 times, 5 minutes each.
8. Place all required materials into a darkroom, turn off the lights, and use infrared illumination.
9. Drain excess buffer from the NC membrane, then add an equal volume (0.125 mL/cm²) of mixed enhanced chemiluminescence (ECL) solution A and B.
10. Place the NC membrane face down and incubate for 3 minutes while cutting the film to size.
11. Turn off the red light and check for visible luminescence; determine the exposure time based on signal intensity.
12. Place the NC membrane face up in the center of the cassette, removing air bubbles and wrinkles in the plastic wrap.
13. Expose the film for 3–5 minutes.
14. Immerse the film in developer solution.
15. Observe the appearance of dark bands.
16. Rinse the film in clean water.
17. Immerse the film in fixing solution for 1–2 minutes to stop the reaction.

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Acrylamide:

• 911990 Acrylamide, 99%, Electrophoresis Grade CAS:79-06-1
• 977281 N,N'-Methylenebisacrylamide, 99%, Electrophoresis Grade CAS:110-26-9

Electrophoresis running buffer：

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10%SDS:

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• 226162 Tris(hydroxymethyl)aminomethane, 99.5%, extra pure CAS:77-86-1
• 978967 Bromophenol Blue, for analysis, Indicator CAS:115-39-9
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• 949300 Methanol, 99.5%, for analysis CAS:67-56-1

TBS-T:

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• 983704 Tween 20 CAS:9005-64-5

Other Reagents：

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• 923740 Isopropanol, 99.7%, for synthesis CAS:67-63-0
• 994220 DNA Marker, Band:100, 250, 500, 750, 1000, 2000 bp

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