Western Blot Protcol

FGI-SOP-2002.8.1

Sample Preparation:

1. Cells (106 cells are homogenized/polytron/sonicate) in 1ml of SolObuffer with protease inhibitor A and B or with phosphatase inhibitors (depending upon experiment).  Tissues can be grinded and then homogenized at 10% w/v in SolObuffer with protease inhibitors.
2. The samples were mixed in SDS-PAGE Laemmli’s buffer and reduced by adding 2.5% BME
3. The cells extracts were heated to 90oC for 3 minutes before applying on SDS-PAGE.
   1. Note:  target proteins that are highly glycosylated/large in size should be avoided freeze thaw cycles or they form oligomers that will not enter in to the gel. 

Western Blot:

1. Prepare SDS gel to the desired percentages (8% for antibodies >50kDa; 10% for antibodies <50kDa but >30kDa; 12% gel for antibodies <30kDa) using 4% gel for stacker.
2. Load the samples (20-65ug protein in 10-25ul volume) into their respective wells; run the gel until dye reaches the bottom. Takes 35-40 minutes for mini gels.
3. Transfer the gel onto nitrocellulose/immobilon (PVDF) membrane in Towbins buffer.
   1. Note:  Immobilon membranes must be wetted in 100% methanol before using for transfer.  For high MW/large glycosylated proteins, reduced the concentration of methanol form 20% to up to 15% and transfer for longer times (plate electrodes: Limit 250mA at 50 volts for 7 hours) the conditions are different for wire electrodes, overnight transfers will be preferred for HMW proteins at low methanol concentrations). 
4. Stain with Ponceaus to assess the quality of transfer and to mark the MW markers on membrane.
5. Place the membrane in blocking buffer (5X DiluObuffer) for 1 hour on shaker at room temperature or overnight at 4oC. 
   1. Note:  If blocking buffer contains Casein then it must be washed with PBS or TBS, this step will increase the signal to noise ratios on the blot. 
6. After blocking, rinse the membrane twice in 1XTBST and wash twice for 5 minutes each time in 1X TBST on shaker. Wash one more time in 1XPBS for 5 minutes on shaker.
7. Dilute the primary antibodies at 1:250-1:1000 in 3ml of antibody dilution buffer (1X DiluObuffer) per 1 cm2 of membranes. Incubate the membranes with primary antibodies for 1 hour.
   1. Note: If the antibody dilution buffer contains milk/BSA or ovalbumin, increase the concentration of primary antibody. The casein/BSA and other proteins will bind Igs and reduced the effective concentration of antibody for blotting.  If the antibody was diluted in DiluObuffer, the remaining antibody can be stored at 4oC for reuse.  
8. After 1 hour, wash 3X with 1X TBST (10 minutes each time).
9. Incubate with appropriate secondary antibody diluted in 1X DiluObuffer (FabGennix Secondary antibody conjugates are diluted 1:15,000-25,000 in DiluOBuffer for optimal signal to noise ratios on blots)
10. After 1 hour, wash 3X with 1X TBST (10 minutes each time).
11. Cover the membranes with chemiluminescent substrate (GlObuffer), acquire images using CCD cooled camera.