SpeedRun One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric)

The SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric) is a complete set of optimized reagents designed for specifically measuring CRISPR-associated protein 9 (Cas9 and dCas9: S. pyogenes) amounts quantitatively using purified Cas9 nuclease or whole cell extracts isolated from tissues and cultured cells of various species. The kit has the following advantages and features:

• Fast: Quick and efficient procedure, which can be finished within 1 hour
• Sensitive: High sensitivity and specificity. The detection limit is as low as 1 ng/well with dynamic range of 2-50 ng/well within the indicated amount range of the whole cell extracts. Only recognizes (S. pyogenes) Cas9 (SpCas9) and dCas9.
• Convenient: The control is conveniently included for the quantification of Cas9 amount
• Flexible: Strip microplate format makes the assay flexible: manual or high-throughput
• Reliable: Simple, reliable, and consistent assay conditions

Background Information
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR-associated protein 9 nuclease) were originally identified in bacteria as a defense mechanism against foreign DNA and have become important tools for targeting and modifying genetic sequences in gene editing and genome engineering applications. The CRISPR/Cas9 system enables sequence-specific cleavage of a targeted genomic locus by delivering RNA-guided Cas9 nuclease and appropriate guide RNAs (gRNA) into cells. A Protospacer Adjacent Motif (PAM) sequence immediately following the specificity sequence is also required for successful Cas9 binding. Monitoring the level of Cas9 editing protein in transfected cells can help assess transfection efficiency and optimize the editing process within the total cell population. Traditional methods such as western blot require electrophoresis and transfer steps, making them more time-consuming and less suitable for high-throughput analysis. To address this need, EpigenTek developed the SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric) for convenient quantitative measurement of Cas9 protein.

Principle & Procedure
The kit is designed for measuring Cas9 levels in whole cell extracts or purified Cas9 proteins from various cells and tissues. In this assay, Cas9 proteins in samples are tightly and stably bound to strip wells. The bound Cas9 proteins are recognized with a detection antibody followed by a color development reagent. The amount of Cas9 is proportional to the absorbance intensity measured and can be quantified by comparison with the Cas9 protein standard.

Starting Materials
Input materials can be purified Cas9 nuclease or whole cell extracts. The amount of whole cell extract for each assay can range from 0.5 to 5 µg, with an optimal range of 1 to 2 µg. The amount of purified protein can range from 1 to 100 ng, with an optimal range of 20 to 50 ng depending on protein purity. The total volume of input material should not exceed 4 µl.