Western Buffer C (for Gialpha2)

For preparing tissues for Western Blot Analysis (e.g., Gialpha2). Use western buffer C to prepare initial dilutions. Sample should be diluted to twice the desired concentration using western buffer A. The final dilution will be 1:1 with western buffer D

To make 100ml
- 40ml of 10% SDS* (FW=288.38), final concentration 4% SDS
- Add 0.0276g of sodium orthovanadate (FW=183.9) to the 20ml SDS; final concentration 1.5mM
- 25ml of 50mM Tris Buffer (PH 7.4); final concentration 12.5mM
- QS to 100 ml with dH2O

*10% SDS: 10G of SDS qs to 100ml with dH2O

For Giα2 western blotting, samples are homogenized in TRIS. The sample is centrifuged, the pellet resuspended in TRIS and protein determination is then made. The samples are centrifuged again and the pellet is resuspended in Western Buffer C. Buffer C is then used to dilute the samples for Western Blot assays. The dilution should be to twice the final desired concentration.

Then, the diluted samples in Western Buffer C and are diluted 1:1 with Western Buffer D to reach the final desired concentration. All samples are now boiled for 5min and loaded on the gel.