In general, high background can be a result of one or more of the following:

(1) Blocking was insufficient. Increase concentration of blocking agent. Increase incubation time with blocking agent. Try alternate blocking agent.

(2) Reagents were too concentrated. Dilute primary antibody, secondary antibody, and/or streptavidin or avidin conjugate. Use recommended substrate concentration.

ELISA-specific suggestions for reducing high background:
-Debris or other component interfered with plate reading. Centrifuge or filter turbid samples before use. Blood-derived samples exhibiting excessive hemolysis or lipemia may interfere with detection and increase background. Use care in pipetting into or out of the wells to avoid air bubbles in the wells (break any air bubbles remaining on plate). Increase wash volumes to more effectively remove residual reagents. Add one or more soaking steps to the plate washing protocol.
-Blocking was insufficient. Increase the concentration of blocking agent. Try alternate blocking agent.

Membrane-bound-specific suggestions for reducing high background:
-Blocking was insufficient. Try alternate blocking agents such as BSA, casein, or nonfat dried milk (nonfat dried milk may contain endogenous biotin or phosphatase activity that may interfere with detection). Increase the concentration of blocking agent.
-Bands or spots appeared in the absence of primary antibody. Dilute the secondary antibody or the streptavidin or avidin conjugate to avoid non-specific interactions. Try alternate reagents. Try alternate detection schemes.
Immunocytochemistry-specific suggestions for reducing high background:
-Blocking was insufficient. Use more blocking agent (such as BSA or control serum).
-Tissues were allowed to dry during incubation. Use humidified chambers for incubation steps.
-Cell or tissue contained large amounts of endogenous biotin (when using avidin/streptavidin system). Block with sequential applications of streptavidin and biotin before using a streptavidin-based system. If endogenous biotin is excessively high, switch to an antibody-based secondary detection system.
-If HRP-based detection is used, remember to inactivate endogenous peroxidase activity in the sample.
-Slide contained excessive adhesive. Reduce the amount of adhesive used.
-Paraffin residue remained on sample. Use more changes of xylene or longer time in xylene.
-Tissue was necrotic. Use alternate tissue, if possible.

Immunofluorescence microscopy-specific suggestions for reducing high background:
-Autofluorescence occurred. Use glass instead of plastic specimen support. Use different coverslip mounting medium. Use another fluorochrome that emits at a different wavelength.
-Filters in microscope were incorrect. Use combinations of excitation and emission filters to select only the wavelengths of interest.
-Dirt on objective. Clean objective.
-Non-specific binding caused by fluorescent dyes can be blocked with Image-iT FX signal enhancer (Cat. No. I36933).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Biotin and derivatives