Western Blotting refers to the electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to sheets of PVDF or nitrocellullose membrane, followed by immunodetection of proteins using antibodies with fluorescent or chemiluminescent detection.

█ Materials:

--Sample (cell lysate or tissue extract)

--Primary antibody against your target protein

--Secondary antibody conjugated to horseradish peroxidase (HRP)

--Blocking buffer (usually 5% non-fat dry milk in TBST)

--TBST buffer (Tris-buffered saline with 0.1% Tween-20)

--Electrophoresis gel (typically a polyacrylamide gel)

--Transfer membrane (nitrocellulose or PVDF)

--Molecular weight markers

--Chemiluminescent substrate (ECL or SuperSignal)

█ Equipment: 

--Electrophoresis apparatus and power supply

--Transfer apparatus (wet or semi-dry)

--Membrane blocking incubator

--Membrane antibody incubation containers

--Chemiluminescence detection system (e.g. X-ray film or digital imager)

█ Protocol:

1) Prepare a polyacrylamide gel appropriate for the size of your protein of interest. Load your protein sample onto the gel and run the gel using an electrophoresis apparatus.

2) Prepare transfer buffer according to the manufacturer's instructions. Set up a transfer apparatus according to the manufacturer's instructions.

3) Transfer the proteins from the gel to a nitrocellulose or PVDF membrane using the transfer apparatus.

4) Block the membrane with blocking solution for 1 hour at room temperature or overnight at 4°C on a rocking platform.

5) Dilute the primary antibody in blocking solution according to the manufacturer's instructions. Incubate the membrane with the primary antibody for 1 hour at room temperature or overnight at 4°C on a rocking platform.

6) Wash the membrane three times with TBST for 5-10 minutes each time.

7) Dilute the secondary antibody conjugated to HRP in blocking solution according to the manufacturer's instructions. Incubate the membrane with the secondary antibody for 1 hour at room temperature on a rocking platform.

8) Wash the membrane three times with TBST for 5-10 minutes each time.

9) Prepare ECL substrate according to the manufacturer's instructions.

10) Incubate the membrane with ECL substrate for a few minutes.

11) Visualize the signal on X-ray film or using an imaging system.