High molecular proteins were difficult to be detected,because it hard to be transmembrane. We suggest that the transmembran should be longer and the voltage should be higher,wet tansmembrane is a good choice too. Besides the sample loading must be as much as possible. As we all known the high molecular bands are tiny in western blotting.
Most of CD antigens molecular weight are over 100KD , belong to high molecular proteins. The high molecular proteins were multi-transmembran, and is immportant for cells.
Use high quality nitrocellulose membrane Pore size 0.2 µm is generally recommended; membranes with a pore size of 0.45 µm are not recommended for proteins smaller than 30 kDa. PVDF membranes may yield higher background than nitrocellulose. Nylon membranes are not recommended for western blotting. Block for 1 hr at room temperature in 5% nonfat dry milk in TBST.
Incubate primary antibody overnight at 4°C in TBST at the recommended dilution with the recommended blocking agent (either 5% BSA or 5% nonfat dry milk). For individual antibodies please consult the product datasheet for recommended dilution buffer.
Washing for less time than the recommended three times 5 min in TBST is common, and can result in high background. We recommend that washes be timed to ensure accuracy. Additionally, low Tween® 20 can contribute to high background; we recommend 0.1% Tween® 20. This applies to washing steps after both primary and secondary antibody incubations.