Not enough antibody is bound to the protein.Īdd a higher concentration of primary antibody Make sure that the isotypes of the primary and secondary are compatible.

Make sure you use a secondary antibody raised against the primary antibody species. The primary antibody and the secondary antibody are not compatible. If only the sample lanes are difficult to see, and the molecular weight ladder is unaffected, this suggests there are issues detecting the protein of interest. This may require some optimization to get right.īands in the sample lanes are faint or have no signal Try imaging the blot again with a longer exposure time. There may not be enough exposure time when imaging the blot. You may have used the wrong filter settings for detection.Įnsure you set the instrument to read the correct wavelengths. Store and handle fluorophores and fluorophore-conjugated antibodies in the dark and minimize light exposure by wrapping the vial in foil. If using fluorescent detection, the fluorophore may have been damaged by too much light exposure. Reagents may have lost activity due to improper storage and handling.Ĭheck the storage instructions for your products on the datasheet. Use fresh, sterile buffer (eg our sterile PBS). The wash or incubation buffer is contaminated with bacteria. Reduce the duration or number of washing steps. Washing with buffer between steps is necessary, but sometimes washing too aggressively can remove detection reagents. If using a PVDF membrane, make sure you pre-soak the membrane in methanol and then in transfer buffer. If the proteins have not transferred effectively, check the transfer was performed in the right direction ( see diagram). Problems with transfer of proteins to the membrane.Ĭheck the transfer was successful using a reversible stain such as Ponceau S before immunostaining.

Western blot ladder but no protein