Please tell us as much information as possible about your experiment ; the conditions, the controls, any troubleshooting tips that you have attempted …. E.g.

• Sample preparation
• Electrophoresis and transfer conditions
• Blocking conditions
• Primary antibody conditions
• Secondary antibody conditions
• Wash steps
• Detection

• DNA detection method (regions amplified in PCR, microarray, direct sequencing)
• DNA purification (decrosslinking conditions, DNA purification method)
• Immunoprecipitation conditions (buffer, amount of detergent, primary antibody manufacturer/dilution, incubation time, matrix for isolating antibody complex)
• Sample preparation (X-ChIP/N-ChIP, cross-linking conditions, DNA fragment size)
• Wash steps

• Amount of protein used
• Blocking conditions
• Detection method
• Primary antibody conditions (diluent/dilution, incubation time/temperature)
• Secondary antibody conditions (diluent/dilution, incubation time/temperature)
• Type of ELISA (e.g. direct, indirect, sandwich ELISA)
• Wash steps
• What did the standard curve look like?

• Blocking/fixation conditions
• Detection and analysis method
• Number of cells used
• Primary antibody conditions (diluent/dilution, incubation time/temperature)
• Secondary antibody conditions (diluent/dilution, incubation time/temperature)
• Wash steps

• Primary antibody conjugation conditions (amount, isotype, buffer, incubation time/temperature)
• Sample preparation, treatment(s) (sample type, method of protein removal)
• Storage conditions of kit components
• Was the exact protocol followed (as supplied with the kit)?
• Which kit component is causing the issue (enzyme/developer/standard)

• Blocking conditions
• Detection method, amplification steps (if any)
• Primary antibody conditions (diluent/dilution, incubation time/temperature)
• Sample fixation, antigen retrieval, permeabilization
• Secondary antibody conditions (diluent/dilution, incubation time/temperature)
• Wash steps

• Amount of protein used
• Detection method
• Matrix used for pull-down
• Primary antibody conditions (diluent/dilution, incubation time/temperature)
• Wash steps
• Western blot conditions

• Amount of protein loaded
• Blocking conditions
• Detection method
• Electrophoresis and transfer conditions
• Primary antibody conditions (diluent/dilution, incubation time/temperature)
• Secondary antibody conditions (diluent/dilution, incubation time/temperature)
• Wash steps

• Blocking conditions, if applicable
• Description of the experiment you are running, with assay conditions (protocol summary, concentrations, incubation conditions)
• Detection method
• Primary antibody conditions (diluent/dilution, incubation time/temperature)
• Sample preparation (amount of protein /cell number /fixation or permeabilization conditions)
• Secondary antibody conditions (diluent/dilution, incubation time/temperature)