I. No signal
1. Ensure enough amounts of antigens are fixed to wells.
2. Decrease the milk percentage in blocking buffer and/or antibody dilution buffer as non-fat dry milk may mask some antigen sites.
3. Increase the concentration of primary antibody &/or secondary antibody.
4. Increase the incubation time for primary antibody and/or secondary antibody incubation.
5. Confirm host species and Ig type of primary antibody as incorrect secondary antibody may be used.
6. Is the antibody correctly stored?

II. High background
1. Increase number of washes in steps to avoid insufficient washing.
2. Increase the blocking time and/or adjust milk concentration to encounter the problem of insufficient blocking.
3. Reduce the concentration of primary antibody or secondary antibody.
4. Check buffers for possible bacterial contaminations.

I. No bands observed/ weak bands observed
1. Increase the amount of total protein (or cell lysate) loaded on gel. Total lysate should be loaded at minimum 100 ug per lane, more is better.
2. Try to use PVDF membrane (or PSQ membrane for low molecular weight proteins) rather than nitrocellulose membrane.
3. Ensure a good transfer between membrane and gel.
4. Decrease the milk percentage in blocking buffer and/or antibody dilution buffer as non-fat dry milk may mask some antigen sites.
5. Increase primary and/or secondary antibody concentration.
6. Extend incubation time for either primary antibody or secondary antibody incubation or both.
7. Reduce the number of washes to minimum in steps to encounter the problem of low protein-antibody binding.
8. Are ECL reagents fresh?
9. Confirm the expression of targeted proteins in cells.
10. Confirm host species and Ig type of primary antibody as incorrect secondary antibody may be used.
11. Is the antibody correctly stored?

II. Non-specific bands/ High background
1. Increase the blocking time and/or adjust milk concentration to encounter the problem of insufficient blocking.
2. Reduce the concentration of primary antibody and/or secondary antibody.
3. Reduce the amount of total protein (or cell lysate) loaded on gel.
4. Increase the number of washes in steps to remove some non-specific binding of primary and/or secondary antibodies.
5. Ensure there is neither aggregation of analyte nor degradation of analyte.
6. Check buffers for possible bacterial contaminations.
7. Reduce film exposure time.

I. No Staining
1. Does your tissue section do present the specific antigen?
2. Deparaffinize sections longer or change fresh xylene solvent as inadequate deparaffinization may be encountered.
3. Increase the concentration of primary and/or secondary antibodies.
4. Increase the incubation time for primary and/or secondary antibodies.
5. Adjust the time of post fixation or try different fixatives by considering improper tissue fixation.
6. Is your antibody properly stored?
7. Confirm host species and Ig type of primary antibody as incorrect secondary antibody may be used.
8. Overstaining/ High background
9. Block endogenous peroxidase activity by 3 % hydrogen peroxide.
10. Increase the blocking time.
11. Reduce the concentration of primary and/or secondary antibodies.
12. Reduce the incubation time for primary and/or secondary antibodies.
13. Increase the number of washes in steps.
14. Avoid samples being dried out.
15. Check buffers for possible bacterial contaminations.

I. No staining/ weak staining
1. Increase the concentration of primary and/or secondary antibodies.
2. Increase the incubation time for primary and/or secondary antibodies.
3. Are proteins expressed in cells?
4. Is your antibody properly stored?
5. Confirm host species and Ig type of primary antibody as incorrect secondary antibody may be used.

II. High background
1. Increase the blocking time.
2. Reduce the concentration of primary and/or secondary antibodies.
3. Reduce the incubation time for primary and/or secondary antibodies.
4. Increase the number of washes in steps.
5. Check buffers for possible bacterial contaminations.