Tests in Pall laboratories have shown that Hammersten grade casein is the most effective blocking agent for all tested membrane types. Hammersten casein was compared to other caseins, casein hydrolysates, non-fat dry milk, gelatin and Bovine Serum Albumin (BSA) for inhibition of binding ¹²⁵I-labeled IgG. 0.5% Hammersten casein produced the largest decrease in binding. Several other caseins also performed well, as did 5% non-fat dry milk. BSA and gelatin are not effective blocking agents for nylon or PVDF membranes, though they will work well on nitrocellulose and styrene microplates.

An experiment was performed to dissect components of Denhardt’s hybridization solution. This is a complex mixture of reagents designed for suppression of non-specific DNA binding to nitrocellulose membranes, containing:

• BSA
• Sheared herring sperm DNA
• Ficoll
• Polyvinylpyrrolidone
• Sodium Doceyl Sulface (SDS)
• Buffer

Each ingredient was tested separately on Pall Nylon 6,6 membranes, which are now more commonly used for DNA detection procedures. BSA and SDS displayed some blocking activity, while the other components (including non-homologous DNA) had little or no effect on non-specific binding to the membrane.

More modern hybridization solutions, such as the buffer recommended for ³²P hybridizations by the FBI, are simplified and eliminate the unnecessary components. Kits for non-radioactive DNA detection generally include casein in the hybridization and protein blocking steps.

Hammersten casein is dissolved in buffer by heating on a stir plate. Maximum solubility is approximately 1% w/v.

Hammersten grade casein can be purchased in the US from Gallard Schlessinger Scientific Supplies, in New York or ICN in California.