Antibodies Other Species / Fungi

Recommended western blot protocol for work with Rnr3 antibodies

1. Run 7.5% SDS-PAGE, run out proteins up to 40 kDa in size.

2. Transfer proteins to a nitrocellulose membrane Protran BA 85, Whatman; 1h RT 300V/300 mA.
Transfer buffer: 50 ml 40xNaCarb, 400 ml Ethanol (95,5%), 1450 ml ddH20.
40xNaCarb: 42.19 g NaHCO3, 36.7 g Na2CO3, dissolve in 1.5 L ddH2O, adjust pH to 9.5 with HCl, bring to 1.8 L and autoclave.

3. Stain with Ponceau S, cut the membrane between 70 and 85 kDa, and wash away the dye with autoclaved ddH2O. Upper part is used for Rnr3 Western Blot.

4. Block membrane with 5% non-fat milk in 1xTBST, 1.5 h RT
10xTBST 1L: 100 ml 1M Tris pH=8.0, 87.66g NaCl, 5 ml Tween20.
Dilute Ab 1:5000 in 5% non-fat milk in 1x TBST 1:5000, incubate ON +4C (can be stored at -20°C and re-used up to 4 times).

5. Wash 3 times in 1xTBST, 10 min each RT

6. Incubate with a secondary Ab 1:5000 in 1xTBST no milk, 1h RT

7. Wash 3 times in 1xTBST, 10 min each RT

8. Develop the membrane.