SimpleChIP® Technical Support
NOTE: For the best possible results, Cell Signaling Technology (CST) strongly recommends using our optimized application-specific protocols for each product. These protocols are the result of extensive in-house validation performed at CST and ensure accurate and reproducible results. Have you consulted our ChIP Troubleshooting Guide before using this technical report?
To ensure a timely solution, please have the information below ready when you contact us. Technical Support can be reached by toll free phone at 877-678-8324, by fax at 978-867-2400 or e-mail at email@example.com.
- Product catalog number (ChIP Kit and/or Antibody).
- Lot number and reference date printed on tube.
- List cell type and treatment used. Adherent or suspension cells?
- Give total number of cells used in the chromatin preparation and explain how cell number was determined.
- Fixation time and formaldehyde concentration.
- Provide nuclease digestion conditions (time, temperature, amount of nuclease added, total volume of digest).
- Time on ice after dissolving pellet in 1x ChIP Buffer.
- Provide sonication conditions (number of pulses, time sonicated each pulse, and time on ice between pulses).
- Sonication model (include probe size and sonication setting).
- Concentration of purified chromatin DNA sample (see Section B of protocol).
Chromatin Immunoprecipitation (ChIP)
- 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.
- Amount of chromatin added to each IP (μg).
- Incubation time of antibodies and chromatin.
- Type of Protein G beads used (magnetic or agarose).
- Incubation time with beads.
- Elution conditions (time and temperature).
- Cross-link reversal conditions (time and temperature).
Detection and Quantification of IP Enrichment
- Method of quantification (standard PCR, quantitative PCR, ChIP-on-chip, ChIP sequencing).
- Number of PCR cycles run and target DNA locus tested.
- Was PCR analysis performed on a serial dilution of the 2% input DNA for the primer set used (described in Section G, Step 1)?
- Was the PCR of the input DNA serial dilution linear using this primer set?
- What was the calculated PCR efficiency for the primer set used (for quantitative PCR users only)?
- 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?
- What was the amount of enrichment of the RPL30 promoter using Normal Rabbit IgG (#2729), as a percent of the total input chromatin?
- 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?
- What was the amount of enrichment of the target locus using Normal Rabbit IgG (#2729), as a percent of the total input chromatin?
- 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?