Western Buffer D (For Giα2 )

Use Western Buffer D for final 1:1 dilution of samples before loading.

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 used to dilute the samples for Western Blot assays. The dilution should be to twice the final dseired concentration.

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.

To make 100ml

• 4g SDS; yields 4% or 40ml of 10% SDS (FW=288.38), final concentration 4% SDS; in some cases may require 10% final concentration of SDS (can use 50ml of 20% SDS or 25ml of 40% SDS)
• 2ml beta -Mercaptoethanol; yields 2%
• 20ml of 100% glycerol; yields 20%
• 1.36gTRIS base (FW=121.1); yields 112.5mM
• Bromophenol blue; yields 0.01%
• add distilled water till 100ml

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

*20% SDS 20G of SDS qs to 100ml with dH2O

*40% SDS 40G of SDS qs to 100ml with dH2O