Drosophila Spike-In Control Kit for CUT&Tag (Mouse) #19629
Product Information
Storage
Product Description
The Drosophila Spike-In Control Kit for CUT&Tag (Mouse) is conveniently designed to provide the reagents required to allow for normalization of variabilities across the entire CUT&Tag experiment using mouse primary antibodies. It starts with the addition of cryopreserved Drosophila spike-in nuclei to cell samples prior to performing the CUT&Tag assay. Like the cells of interest, the spike-in nuclei also bind to the Concanavalin A beads, providing a method to immobilize the spike-in nuclei for subsequent washes and incubations. During the antibody incubation step, an H2Av Mouse Monoclonal Antibody is added to the reaction in addition to the mouse primary antibody against the target of interest. This H2Av Mouse Monoclonal Antibody provides a reliable method to target, digest, and enrich histone H2Av-bound Drosophila chromatin in a consistent way across all samples. This enrichment of H2Av-bound Drosophila chromatin occurs in the same tube as the enrichment of the target of interest from the test cell chromatin, ensuring that the spike-in control nuclei are subjected to all of the same steps and conditions as the test cells. A normalization factor is generated based on the Drosophila chromatin enrichment signal and is applied to the test genome to normalize signals across samples (see Figures 1-3). Thus, this spike-in normalization approach enables reliable and consistent normalization across samples, correcting for variations in starting cell number, technical variation among samples during processing, global histone modification changes, and nonspecific enrichment coming from isotype control antibodies (i.e., nonspecific mouse IgG).
This kit is intended for use only in combination with our CUT&Tag Assay Kit #77552 or other validated in-house CUT&Tag protocols. It includes one 165 µL vial of cryopreserved Drosophila Spike-In Nuclei Control, providing enough nuclei for 24 CUT&Tag reactions using a mouse primary antibody with 100,000–250,000 cells or 1–2.5 mg of tissue per reaction, when used at the recommended volume. Also included is the H2Av Mouse Monoclonal Antibody, which specifically detects the Drosophila histone variant H2Av, showing no cross-reactivity to mammalian histones.
If CUT&Tag assays are performed with rabbit primary antibodies, DO NOT use this kit. Please refer to our Drosophila Spike-In Control Kit for CUT&Tag (Rabbit) #29811 for sample normalizations.
Specificity / Sensitivity
Background
A major challenge in comparing CUT&Tag datasets is being able to account for differences in signal resulting from sample-to-sample variability, such as differences in starting cell number, technical variations among samples during processing, and variations in global histone modifications. The Drosophila Spike-In Control Kit for CUT&Tag (Mouse) provides a complete normalization strategy that allows for normalization of variabilities across the entire experiment (4,5), ensuring a more accurate measurement of protein–DNA interactions between samples and across experiments.
Limited Uses
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