🧪 Western Blot Protocol
Protein Detection and Quantification through Immunoblotting
MATERIALS
· REDI-PRO™ ImmunoBlot
· Filter Paper (Whatman)
· Tweezers
· X-ray film
· X-ray film processor
· Pipette tips
· Pipettor, small volumes
BUFFERS
Blocking Buffer
· 5% non-fat dry milk
· TBST
Wash Buffer (TBST)
· 125 mM NaCl
· 25 mM Tris pH 8.0
· 0.1% Tween-20
Rehydration
- Soak the blot in blocking buffer for 30 minutes prior to use.
Blocking
- Incubate the blot with blocking buffer overnight at 4°C or 2 hours at room temperature with gentle agitation.
- Remove blot from blocking solution.
Primary Antibody Incubation
- Dilute antibody to the recommended dilution in 10mL of blocking buffer.
- Incubate the blot with the primary antibody for one (1) hour at room temperature or overnight at 4°C.
- Wash the blot three (3) times 10 minutes each in washing buffer with gentle agitation.
Secondary Antibody Incubation
- Dilute 1μL anti-rabbit IgG-HRP conjugated secondary (or other appropriate secondary) in 10mL of blocking buffer to make a 1:10000 dilution
- Note: working dilution of secondary can vary from 1:2000 to 1:10000.
- Incubate blot with secondary antibody for one (1) hour at room temperature.
- Wash three (3) times for 10 minutes each in washing buffer with gentle agitation.
Development
- Drain wash buffer
- Add ECL solution (Amersham) per manufacturer directions and develop for 1 minute.
- Drain the fluid.
- Cover the blot in plastic wrap.
- Expose the blot to X-ray film for 1 minute in a dark room.
- If there is no banding, expose the film for 5 minutes, then 30 minutes and up to overnight if the signal is weak.
- If the signal is strong, expose the film for 30 seconds or less.
- Develop the film in an X-ray processor
Notes
- Optimal dilutions should be determined by each laboratory for each antibody.
Reprobing
- Incubate blot for 10 minutes at room temperature in 100mM Glycine, pH 2.5.
- Wash for 10 minutes in DI H2O.
- Redo protocol above.