A Western blot enables the transfer of protein bands from a gel to a membrane followed by probing of the membrane with antibodies to identify specific bands. These instructions are written for use with the Expedeon RunBlue system, but the principle applies to other systems too.
Run and SDS-PAGE gel using pre-stained molecular weight markers. Do not stain your gel.
Prepare two pieces of filter paper and one piece of nitrocellulose / PVDF membrane per gel. Use gloves when handling the membrane (to avoid contamination with extraneous proteins) and take care as it is fragile and expensive.
Place membrane in transfer buffer to equilibrate for ~10mins. When run, do the same with the gel (2-5mins). Also equilibrate 4 black sponges (press down under the surface of the buffer to release air bubbles trapped in the sponges). (When making up transfer buffer from stock solutions, don't forget that you need to add ethanol to the mixture - less toxic than methanol - in order to get efficient transfer)
Meanwhile, place a stir bar in the bottom of the gel running tank (there is a circle to constrain it).
Build up the following from the black side of the transfer case, covering liberally with transfer buffer throughout the process:
2x sponge (The exact amount of sponge will depend on the depth of the construct)t
filter paper
gel )
membrane ) the protein will migrate with negative charge from the gel to the membrane
filter paper
2x sponge
Roll out any air with a glass pasteur pipette as you go along. Make sure you know which way up the gel is, and also how to identify the 'front' of the blot (the side in contact with the gel) afterwards - this might be by cutting a corner off the gel, or by ensuring that you remember the orientation of the molecular weight markers. Don't forget that the transfer process creates a mirror image of what you see when looking down on the gel.
Close the transfer case, seal with the top clips, and place in the gel tank with the black side towards the centre and seal. Place one cool block each side of the central case, then add transfer buffer until the tank is full. Place the tank on a stirrer and blot at 180mA (200V) for 1-1.5hrs.
Prepare 10ml of 10% Marvel, 0.05% Tween in PBS. Place the membrane protein side up and cover with Marvel for a quick initial blot (handle with forceps). Roll up the membrane (around a glass pasteur pipette sometimes helps) to fit into a 50ml falcon tube. Add Marvel mix and incubate on rollers for 1hr (room temperature is fine).
Wash 3x in PBST, then make up a 5ml solution of 1o antibody in PBS. For most antibodies, a dilution of 1 in 1000 is needed (optimise with new antibodies). Incubate on rollers for 1hr.
Wash 3x (for 2-5 min each) in PBST. Add 5ml 2o antibody in PBS (HRP conjugated for film developing, AP conjugated for colour-substrate developing).
(HRP conjugates - borrow the key to the darkroom from the Bayley lab and switch on the developing machine at this stage)
Wash 3x (for 2-5 min each) in PBST.
Mix equal quantities of ECL reagent on cling film (1ml of each will easily develop 1 blot). Place the blot face down in mix and gently rub in solution. Leave 2-5 mins to develop.
Pat the blot dry with blotting paper and place face down on a clean piece of cling film. Reinforce with blotting paper behind and seal the cling film around the whole package.
Take film, developing case, and blot down to the darkroom. Try a number of exposures from 10s upwards to get a blot with clear bands and good signal to noise ratio. If you want to determine the molecular weight of your bands, you will need to use a film-sensitive marker on your blot (or mark the edge of the blot on the film with marker pen). When optimising exposure time using long exposures, don't forget that the signal from the blot will deteriorate with time.
Dissolve one substrate tablet in 10ml water. This is light sensitive, so dissolve quickly and do just before use. Place membrane on a tray and incubate with substrate until all bands have developed sufficiently. Wash with water to remove substrate and stop the reaction. Leave membrane to dry on paper towels. Bands developed in this manner will fade with time, so scan your blot now if you need a permanent record of the experiment.