I. Required material
Reagents
- A primary antibody , labeled or not, against the target molecule
- A secondary antibody in cases of indirect staining with a non labeled primary antibody
- Chemiluminescence solution for western blot
- Transfer buffer (25mM Tris pH8.5, 0.2M Glycine, 20% Methanol)
- Ponseau S solution (0.1% ponceau, 5% acetic acid)
Buffers
- 4x SDS sample buffer (0.25M Tris-HCl pH 6.8, 8% SDS, 30% Glycerol, 0.02% Bromophenol Blue) containing a reducing agent (either 10% B-ME or 0.3M DTT)
- 1 x SDS Running Buffer made from 10x stock (30.3g tris, 144g Glycine, 10g SDS and make up to 1 L with water)
- 10 x TBS (61g Tris, 90g NaCl, pH 7.6)
- Tris, Glycerol, Bromophenol Blue, B-ME, Glycine, SDS
- Methanol, Tris, Glycine, Ponceau, acetic acid
- Tris, NaCl, non-fat milk powder, tween-20
Material
- Pipets and Pipet-aid
- Light sensitive x-ray film
II. Experimentation length
- Heat of samples at 95-100C for 5 minutes
. Electrophoresis of proteins at 100V constant
. Transfer of proteins for 90 minutes at 100V or overnight at +4°C at 20V.
. Incubation of membrane in blocking solution for 1 hour at room temperature
. Incubation of primary antibody at room temperature for 2 hours prepared in 5% milk/TBST
. Incubate membrane with an appropriate secondary antibody (peroxidase conjugated) for 1 hour at room
. Expose membrane to X-ray film for 1 minute to 1 hour, depending on protein signal and chemiluminesence
- TOTAL : 6-8 hours
III. Protocol
- Protein Electrophoresis :
. Prepare cell / tissue lysate(s)
. Make up lysates to a total volume of 10-20 µl with 4x SDS sample buffer
. Prepare SDS-PAGE gel
. Place gel in running chamber and fill with 1 x SDS Running Buffer
. Heat samples at 95-100C for 5 minutes
. Load 5ul of a control pre-stained protein ladder to track sample migration in the first lane of the gel.
. Load boiled samples
. Electrophorese at 100V constant until the blue bromophenol dye has reached the bottom of the gel
- Protein Transfer :
. Place gel and blotting pads in transfer buffer
. Transfer gel to filter paper wetted in transfer buffer
. Assemble transfer sandwich by orientating cathode, filter paper, gel, membrane (nitrocellulose / PVDF), filter paper, anode to allow protein transfer from cathode to anode
. Electrophorese for 90 minutes at 100V or overnight at +4°C at 20V.
- Protein Detection :
. Incubate membrane in blocking solution for 1 hour at room temperature or at +4°C overnight with shaking
. Incubate primary antibody overnight or at room temperature for 2 hours prepared in 5% milk/TBST
. Remove antibody solution and wash membrane three times for 5-15 minutes in TBST
. Incubate membrane with an appropriate secondary antibody (peroxidase conjugated) for 1 hour at room temperature in 5% milk/TBST.
. Remove antibody solution and wash membrane three times for 5-15 minutes in TBST
- Protein Revelation :
. Expose membrane to X-ray film for 1 minute to 1 hour, depending on protein signal and chemiluminesence method
IV. Search engines
- Primary antibodies
- Secondary antibodies
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