Blot membranes protein binding

Western blotting consists of the following steps (Figure 8.11) (1) Proteins are electrophoretically transferred from a gel to a membrane surface. The transferred proteins bind to the surface of the membrane and are immobilized in a... 

Specific detection of nitrocellulose membrane-bound proteins using a conjugated enzyme. (1) Proteins are transferred from electrophoresis gel to nitrocellulose membrane. Blocker proteins bind to unoccupied sites on the membrane. (2) The membrane is incubated with a primary antibody directed against the protein of interest. (3) A secondary antibody is directed against the primary antibody. (4) The second antibody is conjugated with an enzyme to provide a detection mechanism. Substrate solution is added to the blot. The conjugated enzyme (HRP or AP) catalyzes the conversion of substrate (S) to product (P) to form a colored precipitate at the site of the protein-antibody complex. 

Blot membranes consist of nitrocellulose (BA 85 from Schleicher and Schiill), polyvinyliden difluoride (PVDF) (Immobilon from Millipore), positively charged nylon ( nylon) (Zetaprobe from Bio-Rad), or glass fiber coated with polybrene (GF/C from Whatman). The membranes bind the proteins through hydrophobic (nitrocellulose) or hydrophobic and ionic interaction ( nylon, polybrene-coated glass fiber). Even peptides with only 20 amino acids stiU stick to nitrocellulose. 

Compared to the negatively charged nitrocellulose, +nylon membranes bind three to four times more protein per cm (up to 500 pg/cm ) and have better mechanical qualities. With nylon membranes, it is not necessary to add 20% methanol to the blot buffer. This makes the protein transfer (from the gel onto the membrane) quicker and more efficient. The saturation of the free protein binding sites of the nylon membrane is cause for grief (see Section 1.6.2). 

Proteins bind noncovalently to blot membranes. You can take the blotted protein off the membrane (e.g., for proteolytic digestion or for analysis in the MALDITOF). For nitrocellulose, Lui et al. (1996) have systematically examined the interaction of protein and blot membrane. According to them, Zwittergent 3-16 (1% in 100 mM NH4HCO3) removes between 60 and 90% of the blotted protein from the nitrocellulose. The detergent also works with PVDF membranes, albeit not as well. 

---