Chromatin Immunoprecipitation Technical Support

To ensure a timely solution, please have the information below ready when you contact us.

Have you consulted our Chromatin Immunoprecipitation (ChIP) Troubleshooting Guide before using this technical report?

General Questions

1. Product catalog number (ChIP Kit and/or Antibody).
2. Lot number and reference date printed on tube.

Chromatin Preparation

1. List cell type and treatment used. Adherent or suspension cells?
2. Give total number of cells used in the chromatin preparation and explain how cell number was determined.
3. Fixation time and formaldehyde concentration.
4. Provide nuclease digestion conditions (time, temperature, amount of nuclease added, total volume of digest).
5. Time on ice after dissolving pellet in 1x ChIP Buffer.
6. Provide sonication conditions (number of pulses, time sonicated each pulse, and time on ice between pulses).
7. Sonication model (include probe size and sonication setting).
8. Concentration of purified chromatin DNA sample (see Section B of protocol).

Chromatin Immunoprecipitation (ChIP)

1. List antibodies used for IP. Provide amount of antibody used per IP (μg or μl), source of antibody, and if antibody has been validated for IP or ChIP.
2. Amount of chromatin added to each IP (μg).
3. Incubation time of antibodies and chromatin.
4. Type of Protein G beads used (magnetic or agarose).
5. Incubation time with beads.
6. Elution conditions (time and temperature).
7. Cross-link reversal conditions (time and temperature).

Detection and Quantification of IP Enrichment

1. Method of quantification (standard PCR, quantitative PCR, ChIP-on-chip, ChIP sequencing).
2. Number of PCR cycles run and target DNA locus tested.
3. Was PCR analysis performed on a serial dilution of the 2% input DNA for the primer set used (described in Section G, Step 1)?
4. Was the PCR of the input DNA serial dilution linear using this primer set?
5. What was the calculated PCR efficiency for the primer set used (for quantitative PCR users only)?
6. Did the control Histone H3 Antibody (#4620) show enrichment of the control RPL30 promoter? If yes, what was the amount of enrichment, as a percent of the total input chromatin added to the IP reaction?
7. What was the amount of enrichment of the RPL30 promoter using Normal Rabbit IgG (#2729), as a percent of the total input chromatin?
8. Did the control Histone H3 Antibody (#4620) show enrichment of the target locus? If yes, what was the amount of enrichment, as a percent of the total input chromatin?
9. What was the amount of enrichment of the target locus using Normal Rabbit IgG (#2729), as a percent of the total input chromatin?
10. Did the test antibody show enrichment of the target locus. If yes, what was the amount of enrichment as a percent of the total input chromatin?