SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric)
Uses
The SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric) is suitable for specifically measuring CRISPR-associated protein 9 (Cas9 and dCas9: S. pyogenes) amounts quantitatively with use of purified Cas9 nuclease and whole cell extracts isolated from tissues and cultured cells of various species.
Input Material
Input materials should be purified Cas9 nuclease or whole cell extract. The amount of whole cell extract for each assay can be between 0.5 µg and 5 µg with an optimal range of 1 to 2 µg. The amount of purified proteins can be 1 ng to 100 ng with an optimal range of 20-50 ng, depending on the purity of the proteins. Total volume of the input material should not be more than 4 µl.
Whole Cell Extraction
You can use your method of choice for preparing whole cell extracts from the treated (transfected) and untreated (untransfected) samples. EpigenTek also offers a whole cell extraction kit (Cat. #OP-0003) optimized for use with this kit. Whole cell extracts should be stored at -80°C in aliquots until use.
Internal Control
The positive control (C9S) is provided in this kit for the quantification of Cas9 protein. Because content of Cas9 can vary in different samples, it is advised to run replicates for each sample to ensure that the signal generated is validated.
Precautions
To avoid cross-contamination, carefully pipette the sample or solution into the strip wells. Use aerosol-barrier pipette tips and always change pipette tips between liquid transfers. Wear gloves throughout the entire procedure. In case of contact between gloves and sample, change gloves immediately.
Kit Contents
| Component | 96 Assays Cat. #P-7104-96 | Storage Upon Receipt |
|---|---|---|
| WB (10X Wash Buffer) | 14 ml | 4°C |
| HBS (High Binding Solution) | 10 ml | RT |
| C9S (Cas9 Protein Standard, 50 µg/ml)* | 20 µl | -20°C |
| CDA (Cas9 Detection Antibody, 1000 µg/ml)* | 8 µl | 4°C |
| MSI (M-Signal Indicator)* | 16 µl | -20°C |
| ES (Enhancer Solution)* | 16 µl | -20°C |
| DS (Developer Solution) | 10 ml | 4°C |
| SS (Stop Solution) | 10 ml | RT |
| 8-Well Assay Strips (With Frame) | 12 | 4°C |
* For maximum recovery of the products, centrifuge the original vial prior to opening the cap.
Shipping & Storage
The kit is shipped in two parts: the first part at ambient room temperature, and the second and third parts on frozen ice packs to maintain approximately 4°C.
Upon receipt: (1) Store C9S, MSI, and ES at -20°C away from light; (2) Store CDA, WB, DS, and 8-Well Assay Strips at 4°C away from light; (3) Store remaining components (HBS and SS) at room temperature away from light.
Check if WB (10X Wash Buffer) contains salt precipitates before use. If so, warm at room temperature or 37°C and shake the buffer until the salts are re-dissolved.
All components of the kit are stable for 6 months from the date of shipment, when stored properly.
Materials Required But Not Supplied
Principle & Procedure
The SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric) is designed for measuring Cas9 level in whole cell extracts or purified Cas9 proteins from various cells and tissues. In an assay with this kit, the Cas9 proteins in samples are tightly and stably spotted on the strip wells. The bound Cas9 proteins can then be recognized with detection antibody followed by a color development reagent. The ratio of Cas9 is proportional to the intensity of absorbance. The absolute amount of Cas9 can be quantitated by comparing to the Cas9 control.
Starting Materials
For the best results, please read the protocol in its entirety prior to starting your experiment.
Input Amount
The amount of whole cell extracts for each assay can be 0.5 µg to 5 µg with an optimal range of 1-2 µg. The amount of purified proteins can be 1 ng to 100 ng, depending on the purity of the proteins.
Whole Cell Extraction
You can use your method of choice for preparing whole cell extracts. EpigenTek offers a whole cell extraction kit (Cat # OP-0003) optimized for use with this kit.
Whole Cell Extract or Purified Cas9 Protein Storage
Whole cell extract or purified Cas9 proteins should be stored in aliquots at -80°C until use.
Working Buffer and Solution Preparation
Prepare Diluted WB (1X Wash Buffer) by adding 12 ml of WB (10X Wash Buffer) to 108 ml of distilled water.
This Diluted WB can now be stored at 4°C for up to six months.
The anticipated approximate volumes of reagents needed are reflected below for this assay.
| Reagents | 1 well | 8 wells (1 strip) | 16 wells (2 strips) | 48 wells (6 strips) | 96 wells (12 strips) |
|---|---|---|---|---|---|
| Diluted WB | 1 ml | 8 ml | 16 ml | 48 ml | 96 ml |
| HBS | 100 µl | 800 µl | 1600 µl | 4800 µl | 10 ml |
| Cas9 Detection Complex | 50 µl | 400 µl | 800 µl | 2400 µl | 4800 µl |
| DS | 0.1 ml | 0.8 ml | 1.6 ml | 4.8 ml | 9.6 ml |
| SS | 0.1 ml | 0.8 ml | 1.6 ml | 4.8 ml | 9.6 ml |
| C9S | N/A | N/A | Optional | 12 µl | 12-20 µl |
Standard or Positive Control (C9S) Curve Preparation
Suggested Standard Curve Preparation: Prepare 6 concentrations by diluting the 50 ng/µl of C9S (Cas9 Protein Standard) with HBS (High Binding Solution) into final concentrations of 1, 2, 5, 10, 20 and 50 ng/µl according to the following dilution chart:
| Tube | C9S (50 ng/µl) | HBS | Resulting C9S Concentration |
|---|---|---|---|
| 1 | 0.5 µl | 24.5 µl | 1 ng/µl |
| 2 | 1.0 µl | 24.0 µl | 2 ng/µl |
| 3 | 1.0 µl | 9.0 µl | 5 ng/µl |
| 4 | 1.0 µl | 4.0 µl | 10 ng/µl |
| 5 | 2.0 µl | 3.0 µl | 20 ng/µl |
| 6 | 4.0 µl | 0.0 µl | 50 ng/µl |
Keep each of diluted solutions, except Diluted WB (1X Wash Buffer), on ice until use. Any remaining diluted solutions other than Diluted WB should be discarded if not used within the same day.
Cas9 Binding Strip Well Setup
Review the configuration of the strip-well plate setup for standard curve preparation strips, showing strip 1 to 6 (7 through 12 can be configured as Sample). The controls and samples can be measured in duplicate, loaded vertically instead of horizontally.
| Well # | Strip 1 | Strip 2 | Strip 3 | Strip 4 | Strip 5 | Strip 6 |
|---|---|---|---|---|---|---|
| A | Blank | C9S 10 ng/well | Sample 2 | Sample 6 | Sample 10 | Sample 14 |
| B | Blank | C9S 10 ng/well | Sample 2 | Sample 6 | Sample 10 | Sample 14 |
| C | C9S 1 ng/well | C9S 20 ng/well | Sample 3 | Sample 7 | Sample 11 | Sample 15 |
| D | C9S 1 ng/well | C9S 20 ng/well | Sample 3 | Sample 7 | Sample 11 | Sample 15 |
| E | C9S 2 ng/well | C9S 50 ng/well | Sample 4 | Sample 8 | Sample 12 | Sample 16 |
| F | C9S 2 ng/well | C9S 50 ng/well | Sample 4 | Sample 8 | Sample 12 | Sample 16 |
| G | C9S 5 ng/well | Sample 1 | Sample 5 | Sample 9 | Sample 13 | Sample 17 |
| H | C9S 5 ng/well | Sample 1 | Sample 5 | Sample 9 | Sample 13 | Sample 17 |
Cas9 Binding
(1) Follow the suggested well setup diagram under "Cas9 Binding Strip Well Setup"; (2) It is recommended to use 1 µg to 2 µg of whole cell extract per well or 20 ng to 50 ng of purified protein per well.
Cas9 Level Detection and Signal Measurement
The color development time may vary from 1-10 minutes based on the speed of color change, but is typically 3-4 minutes.
Cas9 Calculation
Quantification: To quantify the amount of Cas9 using an accurate calculation, first generate a standard curve and plot the OD values (blank-subtracted) versus the amount of C9S at each concentration point. Next, determine the slope (OD/ng) of the standard curve using linear regression (Microsoft Excel's linear regression functions are suitable for such calculation). Use the most linear part of the standard curve (at least 4 concentration points, including blank point) for optimal slope calculation. Now calculate the amount and percentage of Cas9 in your sample using the following formulas:
S is the amount of input sample protein in ng.
In the event that the standard curve is flat due to (1) high ODs starting from the lowest C9S concentration point or (2) a saturated signal intensity at high C9S concentrations from extended color development time, the Cas9 amount can be calculated with non-linear regression, such as logarithmic or polynomial second order regression (see the Appendix section).
A Brief Overview
The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR associated system or CRISPR associated protein 9 nuclease) found in bacteria to work as a defense mechanism against foreign DNA has proven to be an invaluable tool to target and modify a genetic sequence in gene editing and genome engineering applications. The system, known as CRISPR/Cas9, allows for sequence-specific cleavage of a targeted genomic locus by delivering the RNA-guided Cas9 nuclease and appropriate guide RNAs (gRNA) into a cell. In addition, Protospacer Adjacent Motif (PAM) sequence immediately following the specificity sequence is necessary for successful binding of the Cas9 nuclease. It is important and critical to monitor the level of Cas9 editing protein or track the Cas9 editing protein in transfected cells, as it will tell transfection efficiency and optimize the editing process in the total cell population.
Western blot is currently the most prominent assay technique for measuring the expression or amount of Cas9 protein. Yet this traditional method requires electrophoresis and transfer processes, which make the assay inconvenient, time consuming, and low throughput. The SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric) addresses these problems by using a unique procedure to measure the amount of Cas9 proteins. The kit has the following features:
- Quick and efficient procedure, which can be finished within 1 hour.
- 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 Cas9 (S. pyogenes).
- The control is conveniently included for the quantification of Cas9 amount.
- Strip microplate format makes the assay flexible: manual or high throughput.
- Simple, reliable, and consistent assay conditions.
General Product Information
Quality Control: Each lot of SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric) is tested against predetermined specifications to ensure consistent product quality. EpigenTek guarantees the performance of all products in the manner described in our product instructions.
Product Warranty: If this product does not meet your expectations, simply contact our technical support unit or your regional distributor. We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques.
Safety: Suitable lab coat, disposable gloves, and proper eye protection are required when working with this product.
Product Updates: EpigenTek reserves the right to change or modify any product to enhance its performance and design. The information in this User Guide is subject to change at any time without notice. Be sure to use the latest User Guide for this kit, which can be accessed online at www.epigentek.com/datasheet.
Usage Limitation: The SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric) is for research use only and is not intended for diagnostic or therapeutic applications.
Intellectual Property: The SpeedRun™ One-Hour CRISPR/Cas9 ELISA Kit (Colorimetric) and methods of use contain proprietary technologies by EpigenTek.
Troubleshooting
| Problem | Possible Cause | Suggestion |
|---|---|---|
| No signal or weak signal in both the standard and sample wells | Reagents are added incorrectly. | Check if reagents are added in the proper order with the right amount, and if any steps in the protocol may have been omitted by mistake. |
| The sample protein and standard are not properly bound to the wells. | Ensure that (1) the sample protein and C9S (Cas9 Protein Standard) are added into the wells; (2) the wells are completely covered with sufficient HBS (High Binding Solution); and (3) binding time is sufficient (25 min). | |
| Incubation time and temperature are incorrect. | Ensure the incubation time and temperature described in the protocol are followed correctly. | |
| Incorrect absorbance reading. | Check if the appropriate absorbance wavelength (450 nm filter) is used. | |
| Kit was not stored or handled properly. | Ensure all components of the kit were stored at the appropriate temperatures and the caps are tightly closed after each opening or use. | |
| No signal or weak signal only in the standard curve wells | The standard amount is insufficiently added to the well in Step 4d. | Ensure a sufficient amount of standard is added. |
| The standard is degraded due to improper storage conditions. | Follow the Shipping & Storage guidance of this User Guide for storage of C9S (Cas9 Protein Standard). | |
| High background present in the blank wells | Insufficient washing of wells. | Check if washing at Step 5c is performed according to the protocol. |
| Contaminated by sample or standard. | Ensure the well is not contaminated from adding sample or standard accidentally or from using contaminated tips. | |
| Over development of color. | Decrease the development time in Step 5d before adding SS (Stop Solution) in Step 5e. | |
| No signal or weak signal only in sample wells | Protein sample is not properly extracted or purified. | Ensure your protocol is suitable for Cas9 protein extraction. For the best results, it is advised to use EpigenTek's Whole Cell Extraction Kit (Cat # OP-0003). |
| Sample amount added into the wells is insufficient. | Ensure a sufficient amount of purified proteins or whole cell extracts is used as indicated in Step 4e. The sample can be titrated to determine the optimal amount to use in the assay. | |
| Sample was not stored properly or has been stored for too long. | Ensure sample is stored in aliquots at -80°C. Avoid repeated freezing/thawing. | |
| Uneven color development | Insufficient wash of the wells. | Ensure the wells are washed according to the guidance of washing and residual wash buffer is removed as much as possible. |
| Delayed color development or delayed stopping of color development in the wells. | Ensure color development and stop solutions are added sequentially and consistent with the order you added the other reagents (e.g., from well A to H or from well 1 to 12). |
Appendix
Method 1: Target Calculation Using Logarithmic Regression
Use this method when the standard curve is flat due to high ODs starting from the lowest standard concentration.
Here, X = target level represented on the standard curve X-axis; Y = Sample OD; a is the regression coefficient and b is the Y-intercept.
*Microsoft Excel's logarithmic regression function can be used for easy and convenient calculation.
Here, S is the amount of input sample in ng.
Example Calculation
Data was graphed using a Standard Scatter (XY) Chart in Microsoft Excel. In the figure above, a = 0.2508, b = 1.1329, Y = 1.274. Input sample amount was 100 ng.
Target percentage of the sample was calculated accordingly:
Method 2: Target Calculation Using Polynomial Second Order Regression
Use this method when the standard curve is flat due to a saturated signal intensity at high standard concentrations.
Here, X = target level represented on the standard curve X-axis; Y = (sample OD value - negative control or blank OD) represented on the standard curve Y-axis; a and b are regression coefficients.
*Microsoft Excel's polynomial regression function can be used for easy and convenient calculation.
Here, S is the amount of input sample in ng.
Example Calculation
Data was graphed using a Standard Scatter (XY) Chart in Microsoft Excel. In the figure above, a = -0.0865, b = 0.6373, Y = (0.621 - 0.060) = 0.561. Input sample amount was 100 ng.
Target percentage of the sample was calculated accordingly:
If the background-corrected sample OD is less than zero or falls outside the measurable range of the standard curve, the target level should not be calculated by extrapolation. The sample should be considered below the quantifiable range or re-assayed at an appropriate dilution, as applicable.