ANDC DU/Biology Protocols/Western blotting

To perform Western Blotting of proteins from SDS-Polyacrylamide gel

Materials and Reagents :

Western Blot Apparatus consisting of gel holder, sponge and transfer tank. Power pack and electric leads Slab gel containing separated proteins ( SDS – PAGE gel is used ) Nitrocellulose sheet cut to the size of the gel Whatman 3 mm paper cut to the size of the gel Western Transfer buffer: Glycine 57.6 g Tris Base 12.0 g SDS 3.0 g Methanol 800 ml H2O to 4 liters

Principle:

The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. The western blot (alternately, immunoblot) is a method of detecting specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein

Procedure:

 The gel after electrophoresis should be taken marked by making a nick at the one corners. It should not be stained.

 The gel should then be placed in transfer buffer for equilibration

 Take a nitrocellulose sheet and cut to the size of the gel.

 Dip the nitrocellulose sheet in the transfer buffer carefully and leave in it for 30 min.

 Soak a sponge n the transfer buffer and then place wet sponge on the gel holder

 Place a Whattman paper on the sponge

 The equilibrated gel should then be placed on the whattman paper avoiding trapping of the air bubbles.

 The membrane should then be placed carefully with shining surface towards the gel. Roll a sterile 1 ml pippette on the gel to ensure good contact between the gel and membrane.

 Complete the sandwich by placing wet Whatman 3mm paper over the membrane and a second sponge on the filter paper.

 Close the complete set-up and place in a transfer tank containing sufficient transfer buffer to completely cover the blot.

 Connect the apparatus to the powersupply and run for 5h at 60V.

 Lift the membrane from the gel once the transfer is complete.

 The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie or Ponceau S dyes. Coomassie is the more sensitive of the two, although Ponceau S's water solubility makes it easier to subsequently destain and probe the membrane as described below.

Discussion:

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, and a stack of tissue papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this "blotting" process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings

Medical diagnostic applications of western blotting

• The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.
• A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').
• Some forms of Lyme disease testing employ Western blotting.

Precautions:

All the chemicals and distilled water should be of high quality.

Acrylamide is neurotoxic and should be handled with care.

Donot touch the transfer chamber or wires while blotting is in progress.

Extreme care should be taken while placing the membrane on the gel

Work in progress, expect frequent changes. Help and feedback is welcome. See discussion page.