Trouble Shooting Tips
Having trouble with an experiment? Use our checklists to help identify the problem. We have many years experience of doing antibody-related experiments and are delighted to pass that knowledge on to you.
- Western blotting
I. No signal
- Ensure enough amounts of antigens are fixed to wells.
- Decrease the milk percentage in blocking buffer and/or antibody dilution buffer as non-fat dry milk may mask some antigen sites.
- Increase the concentration of primary antibody &/or secondary antibody.
- Increase the incubation time for primary antibody and/or secondary antibody incubation.
- Confirm host species and Ig type of primary antibody as incorrect secondary antibody may be used.
- Is the antibody correctly stored?
II. High background
- Increase number of washes in steps to avoid insufficient washing.
- Increase the blocking time and/or adjust milk concentration to encounter the problem of insufficient blocking.
- Reduce the concentration of primary antibody or secondary antibody.
- Check buffers for possible bacterial contaminations.
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I. No bands observed/ weak bands observed
- 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
- Try to use PVDF membrane (or PSQ membrane for low molecular weight proteins) rather than nitrocellulose membrane.
- Ensure a good transfer between membrane and gel.
- Increase primary and/or secondary antibody concentration.
- Extend incubation time for either primary antibody or secondary antibody incubation or both.
- Reduce the number of washes to minimum in steps to encounter the problem of low protein-antibody binding.
- Are ECL reagents fresh?
- Confirm the expression of targeted proteins in cells.
II. Non-specific bands/ High background
- Reduce the concentration of primary antibody and/or secondary antibody.
- Reduce the amount of total protein (or cell lysate) loaded on gel.
- Increase the number of washes in steps to remove some non-specific binding of primary and/or secondary antibodies.
- Ensure there is neither aggregation of analyte nor degradation of analyte.
- Reduce film exposure time.
I. No Staining
- Does your tissue section do present the specific antigen?
- Deparaffinize sections longer or change fresh xylene solvent as inadequate deparaffinization may be encountered.
- Increase the concentration of primary and/or secondary antibodies.
- Increase the incubation time for primary and/or secondary antibodies.
- Adjust the time of post fixation or try different fixatives by considering improper tissue fixation.
- Is your antibody properly stored?
II. Overstaining/ High background
- Block endogenous peroxidase activity by 3 % hydrogen peroxide.
- Increase the blocking time.
- Reduce the concentration of primary and/or secondary antibodies.
- Reduce the incubation time for primary and/or secondary antibodies.
- Increase the number of washes in steps.
- Avoid samples being dried out.
I. No staining/ weak staining
- Are proteins expressed in cells?
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