Modified Histone H3 Multiplex Assay Kit

Results Summary

The kit supposes to measure the percentage of different histone modification of H3. After conducting the assay, I found that some of the modification is more than 100%. Obviously, such a result doesn’t make sense. so I contacted Epigentek. I also measured the H3 level using another kit (P-3091-96). This kit gave me different result values from EpiQui Circulating total Histone kit (P-3106-96). Still, the percentage of modifications didn’t make sense. Blank and positive controls performed satisfactorily. I had long conversations with technical support. At the bottom line, I learned that 1. According to technical support, I performed the assays “somehow” incorrectly though they are unable to specify the point 2. They cannot explain significant differences in serum level of H3 between two different kits (P-3091-96 vs. (P-3106-96) 3. The kit P-3106-96 was NEVER validated for human serum samples. The company tested the kit by using FBS and histone from cells line. I am not sure how such a condition resembles human serum. This is important since the part of P-3091-96 measuring modification level may non-specifically bind molecule – a potential explanation of over 100% histone modification 4. The manual contains errors. 5. It took over a month to get some response from tech support. Please see their responses verbatim: “Based on the data you provided, the ODs for the blank is quite low, and the ODs for standard control is 100 fold higher than that for blank. These also indicate the kit P-3106 works properly. The question is that the total H3 ODs measured with kit P-3106 are quite low, causing a percentage of some H3 modifications to be more than 100%, and total H3 ODs measured with kit P-3091 is sufficiently high. It is somewhat difficult to explain as the antibody coated on total H3 wells for P-3106 is the exactly same as that for P-3091 and re-tests show that the total H3 ODs measured with both kits are quite similar. However, as ODs for modified H3 seem in the reasonable range and high ODs could be seen as part of modified H3 (ex: 0.459 for H3K27m2 for sample 7d and 0.411 for H3k36m3 for sample t7d), the ODs measured with kit P-3091 would be good to use for total H3 amount calculation, so that the relative comparison between different modified H3 patterns can be achieved with the already generated data.” 6. Solution suggested was to choose the total H3 levels from another kit because “as (1) The signals measured with total H3 P-3091 kit is reasonable and obviously there is a problem with your total H3 signals obtained with P-3106 kit; (2) With the same samples, using a kit for total H3 quantification and then for normalizing % H3 modifications measured with a histone modification kit is widely acceptable, and this application is indicated not only by us but also by other companies; and (3) As you express that you lost samples, the solution we provided allows you to use the already generated data. Otherwise, the solution is to generate new samples and repeat the assay by purchasing a new kit.” I challenged the statement as pick and choose. The company did not return the comment if there is non-sensitive binding fro human serum for serum modification. 7. The kit is prohibitively expensive. You can only run two samples. 8. I requested refund or credit. So far company did nothing in that respect except asking me to remove my comment to the kit from their website.