You shall use skim milk powder free of fat! Alternatively, you may use 3% Bovine serum albumin or Gelatin. It shall work. Don't was the membrane after BDA or Gelatin treatment. As no protein bands except protein markers appeared, it seems your test sample has already degraded. Electrobloting of proteins on membrane may be checked with Ponseau Red dye which is reversible.

• I completely agree with Sham
• I think your milk powder is full of impurities !!
• You can use skimmed milk powder from a supermarket (fat free !!) to save money but you are better off using commercial lab grade 3% BSA for blocking your membrane
• It is curious that you have detected none of your proteins: Poor blocking if any thing would result in non specific signal, not no signal at all
• Have you checked the protein on your membrane by staining with Ponceau S before washing with water and PBS and then blocking (with skimmed milk powder or preferably 3% BSA) ?
• How long did you perform your tranfer for ?
• What do the manufacturers recommend for dilution o your primary antibody and how are you diluting it ? Are you adding your fatty mixture to PBS to dilute your antibodies ?
• Have you tried probing your membrane with a housekeeper like B tubulin or B actin ?

Hi,

I think skimmed milk(non-fat) is being recommended so as to effectively block non-specific binding except phosphorylated proteins on the membrane which you can use 3% BSA in TBS-T.Peradventure you use fatty milk, it could lead to production of so many artifacts on the membrane.Also using non-fat milk helps to give good background and prevent antigen recognition by primary antibody resulting in weaker signal.

However, i will suggest that you should first confirm whether your protein is transferred or not by staining your membrane with Ponceau S as stated by other colleagues.Thereafter, you can proceed to blocking by strictly using non-fat milk to block in TBS-T.

I hope this will be useful to you

Best of luck!

Wh