Western Blot Protocol

Western Blot Introduction

Western Blotting is an analytical Immunoblotting technique to detect specific proteins in a cell extract or tissue homogenate. Western Blotting relies on the specific binding between the protein-of-interest and an antibody raised against this particular protein.

Step-by-step western blot protocol

All steps are carried out at room temperature unless otherwise indicated.

SDS PAGE

• Construct an SDS-PAGE gel according to the molecular weight (MW) of your target protein.

• Prepare samples in microfuge tubes. Add 4X SDS Sample Buffer, so the total protein amount is 30–50 μg per sample (according to the protein amount measured by Bradford or BCA protein assay).

• Flick microfuge tubes to mix samples, and then heat to 95-100°C for 5 minutes.

• Set up electrophoresis apparatus and immerse in a 1x Running Buffer. Remove gel combs and cleanse wells of any residual stacking gel.

• Load samples and protein markers onto the gel. Set electrophoresis power pack to 80V (through the stacking gel) before increasing it to 120V when the protein front reaches the separation gel.

Protein transfer

• PVDF membranes (or PSQ membranes with 0.22 μm micropores when the MW of the target is <30 kDa) are strongly recommended. Soak membranes in methanol for 30 seconds before moving to transfer buffer. Soak the filter papers and sponges in the transfer buffer.

• Sequentially assemble the transfer. In a shallow tray, open the transfer cassette. Put a well-soaked sponge pad on the black transfer cassette. Apply the wetted filter paper to the sponge pad. Next, carefully place the gel on the filter paper and eliminate any air bubbles. Lay the PVDF membrane on the top of the gel and again eliminate any air bubbles. Apply the wetted filter paper to the PVDF membrane and another well-soaked sponge pad. Close the transfer cassette. Apply semi-dry or wet transfer systems according to the manufacturer’s instructions.

Immunoblotting

• After transfer, wash the membrane twice with distilled water, and use a pencil and mark bands of the MW ladder on the membrane. If desired, stain the membrane with commercial Ponceau red solution for 1 min to visualize protein bands, then wash any Ponceau red staining with copious amounts of 1x TBST.

• Block with 1x TBST containing (2-5%) nonfat dry milk (or 1-5% BSA for the detection of phospho-epitope antibodies) with constant rocking for 1 hour or overnight at 4°C.

• Dilute primary antibody in blocking solution with a starting dilution ratio of 1:1000. (Optimal dilutions should be determined experimentally.) Incubate the membrane with primary antibody for 1 hour at room temperature or overnight at 4°C.

• Wash membrane three times with 1x TBST for 10 minutes each.

• Incubate the membrane with a suitable HRP-conjugated secondary antibody (recognizing the primary antibody's host species), diluted at 1:5000–1:50000 in blocking solution. Incubate for 1 hour with constant rocking.

• Wash membrane three times with 1x TBST for 10 minutes each.

Signal detection

• Prepare an ECL substrate according to the manufacturer’s instructions.

• Incubate the membrane completely with the substrate for 1–5 minutes (adjust the time for more sensitive ECL substrates, e.g., SuperSignal West Femto Chemiluminescent Substrate [Pierce]).

• Expose the membrane to autoradiography film in a dark room or read using a chemiluminescence imaging system.

Line up the developed film in the correct orientation to the blot and mark the bands of the MW ladder directly onto the film. It is also advised to add notes such as lane content, film exposure time, and ECL properties