SpeedRun™ One-Hour MBD1 ELISA Kit (Colorimetric)
Uses
The SpeedRun™ One-Hour MBD1 ELISA Kit (Colorimetric) is suitable for measuring the amount of MBD1 using nuclear extracts or purified MBD1 proteins from a broad range of species such as human, mouse and rabbit, in a variety of forms including, but not limited to, cultured cells and fresh/frozen tissues.
Starting Materials
Input materials can be nuclear extracts or purified MBD1 proteins. The amount of nuclear extracts for each assay can be 1 µg to 2 µg. The amount of purified proteins can be 1 ng to 50 ng with an optimal range of 10-20 ng, depending on the purity of the proteins.
Internal Control
The MBD1 assay standard (MBD1 recombinant protein) is provided in this kit for the quantification of MBD1 amount. Because MBD1 level can vary from tissue to tissue and from normal and diseased states, it is advised to run replicate samples to ensure that the generated signal 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-7106-96 | Storage Upon Receipt |
|---|---|---|
| WB (10X Wash Buffer) | 14 ml | 4°C |
| HBS (High Binding Solution) | 10 ml | RT |
| M1S (MBD1 Protein Standard, 20 µg/ml)* | 20 µl | -20°C |
| M1A (MBD1 Capture Antibody, 1000 µg/ml)* | 8 µl | -20°C |
| RSI (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 part on frozen ice packs to maintain approximately 4°C. Upon receipt: (1) Store M1S, M1A, RSI, and ES at -20°C away from light; (2) Store 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 MBD1 ELISA Kit (Colorimetric) contains all reagents necessary for the quantification of MBD1 levels. In this assay, MBD1 in the extracts is bound to strip-wells with a high binding buffer and is detected using MBD1 Detection Complex and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The MBD1 level is proportional to the OD intensity measured.
Starting Materials
For the best results, please read the protocol in its entirety prior to starting your experiment.
Input Amount
The amount of nuclear extracts for each assay can be 1-2 µg. The optimal amount of purified proteins can be 10 ng to 20 ng, depending on the purity of the proteins.
Nuclear Extraction
You can use your method of choice for preparing nuclear extracts. EpigenTek offers a nuclear extraction kit (Cat # OP-0002) optimized for use with this kit.
Nuclear Extract or Purified MBD1 Protein Storage
Nuclear extract or purified MBD1 proteins should be stored in aliquots at -80°C until use.
1. 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 | 9600 µl |
| MBD1 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 |
| M1S | N/A | N/A | Optional | 10 µl | 10-20 µl |
2. Standard or Positive Control (M1S) Curve Preparation
Suggested Standard Curve Preparation: Prepare six concentrations in 0.2 ml M1SR tubes by combining the 20 ng/µl M1S (MBD1 Protein Standard) with HBS (High Binding Solution) into final concentrations of 0.5, 1, 2, 5, 10, and 20 ng/µl according to the following dilution chart:
| Tube | M1S (20 ng/µl) | HBS | Resulting M1S Concentration |
|---|---|---|---|
| 1 | 0.5 µl | 19.5 µl | 0.5 ng/µl |
| 2 | 1.0 µl | 19.0 µl | 1 ng/µl |
| 3 | 1.0 µl | 9.0 µl | 2 ng/µl |
| 4 | 1.0 µl | 3.0 µl | 5 ng/µl |
| 5 | 2.0 µl | 2.0 µl | 10 ng/µl |
| 6 | 4.0 µl | 0.0 µl | 20 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.
3. MBD1 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 | M1S 5 ng/well | Sample 2 | Sample 6 | Sample 10 | Sample 14 |
| B | Blank | M1S 5 ng/well | Sample 2 | Sample 6 | Sample 10 | Sample 14 |
| C | M1S 0.5 ng/well | M1S 10 ng/well | Sample 3 | Sample 7 | Sample 11 | Sample 15 |
| D | M1S 0.5 ng/well | M1S 10 ng/well | Sample 3 | Sample 7 | Sample 11 | Sample 15 |
| E | M1S 1 ng/well | M1S 20 ng/well | Sample 4 | Sample 8 | Sample 12 | Sample 16 |
| F | M1S 1 ng/well | M1S 20 ng/well | Sample 4 | Sample 8 | Sample 12 | Sample 16 |
| G | M1S 2 ng/well | Sample 1 | Sample 5 | Sample 9 | Sample 13 | Sample 17 |
| H | M1S 2 ng/well | Sample 1 | Sample 5 | Sample 9 | Sample 13 | Sample 17 |
4. MBD1 Binding
Predetermine the number of strip wells required for your experiment. Carefully remove un-needed strip wells from the plate frame and place them back in the bag (seal the bag tightly and store at 4°C).
Add 80 µl of HBS (High Binding Solution) to each well.
Blank Wells: Add 1 µl of HBS to each blank well.
Standard Wells: Add 1 µl of Diluted M1S (MBD1 Protein Standard) to each standard well (see the designated wells depicted in the Table under "MBD1 Binding Strip Well Setup" above).
Sample Wells: Add 2 to 4 µl (1-2 µg) of nuclear extracts or purified MBD1 proteins (10-20 ng) to each sample well.
Mix solution by gently tilting from side to side or shaking the plate several times.
(1) Follow the suggested well setup diagram under "MBD1 Binding Strip Well Setup"; (2) It is recommended to use 1 µg to 2 µg of nuclear extract per well or 10 ng to 20 ng of purified protein per well.
Tightly cover strip plate with Parafilm M or aluminum foil to avoid evaporation and incubate at 37°C for 25 minutes.
During the last 10 minutes of sample incubation, prepare the MBD1 Detection Complex Solution: In each 1 ml of Diluted WB add 1 µl of M1A (MBD1 Capture Antibody), mix and then add 1 µl of RSI (Signal Indicator) and 1 µl of ES (Enhancer Solution). Mix well.
Remove the reaction solution from each well. Wash each well one time with 150 µl of the Diluted WB (1X Wash Buffer).
5. MBD1 Level Detection and Signal Measurement
Add 50 µl of the MBD1 Detection Complex Solution to each well, then cover and incubate at room temperature for 25 minutes.
Remove the MBD1 Detection Complex Solution from each well.
Wash each well with 150 µl of the Diluted WB (1X Wash Buffer) each time for four times.
Add 100 µl of DS (Developer Solution) to each well in a column, not row, simultaneously in a vertical fashion with a multi-channel pipette so that replicates are developed at the same time. Gently shake the plate against a flat surface for 5-10 seconds and incubate at room temperature for 3-4 minutes. Monitor color development in the sample wells and control wells. After a few minutes, the DS (Developer Solution) will turn blue in the presence of sufficient bound MBD1. The color in the blank wells will remain generally unchanged or slightly changed.
When the color in the 20 ng/µl M1S wells turns deep blue, stop the enzyme reaction by adding 100 µl of SS (Stop Solution) to each well in the same order as Step 5d. Mix the solution by gently shaking the plate against a flat surface and wait 3-4 minutes to allow the color reaction to be completely stopped. The color will change to yellow after adding SS (Stop Solution) and the absorbance should be read on a microplate reader at 450 nm within 2-15 minutes.
The color development time may vary from 1-10 minutes based on the speed of color change, but is typically 3-4 minutes.
6. MBD1 Calculation
Quantification: To quantify the amount of MBD1 using an accurate calculation, first generate a standard curve and plot the OD values (blank-subtracted) versus the amount of M1S 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 MBD1 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 M1S concentration point or (2) a saturated signal intensity at high M1S concentrations from extended color development time, the MBD1 amount can be calculated with non-linear regression, such as logarithmic or polynomial second order regression (see the Appendix section).
A Brief Overview
MBD1 (methyl-CpG-binding domain protein 1) is a member of the MBD protein family. MBD1 selectively binds to methylated DNA and suppresses transcription from a methylated target gene through recruiting transcriptional repressor complexes. As an epigenetic reader of methylated CpG DNA, MBD1 is a novel target that has been accredited for cancer therapy. Some evidence has also linked this protein to neurodevelopment. The binding of MBD1 to methylated DNA may be affected by MBD1 mutation and biochemical or pharmacological intervention. Selective and potent MBD1 inhibition can induce gene re-expression, as seen with genetic disruption of MBD1 in cancer cells. Thus, detecting level of MBD1 is important in elucidating mechanisms of epigenetic regulation of gene activation and silencing, as well as benefiting cancer and neuro disease therapeutics.
To address this issue, EpigenTek developed and offers the SpeedRun™ One-Hour MBD1 ELISA Kit (Colorimetric). The kit has the following advantages and features:
- Quick: Colorimetric assay completed within 1 hour
- Versatile: Works with cell/tissue extracts or purified MBD1 proteins.
- Sensitive: Limit of detection can be as low as 1 ng of MBD1 proteins
- Convenient: Includes MBD1 protein standard for quantification
- Flexible: Allows manual or high-throughput analysis with strip microplate format
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 M1S (MBD1 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 M1S (MBD1 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 MBD1 protein extraction. For the best results, it is advised to use EpigenTek's Nuclear Extraction Kit (Cat # OP-0002). |
| Sample amount added into the wells is insufficient. | Ensure a sufficient amount of purified proteins or nuclear 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.
- Plot the average OD value on the Y-axis versus the known target level of each standard point on the X-axis.
- Graph the logarithmic curve* (also see "Example Calculation" below) and obtain the logarithmic regression equation:
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.
- Calculate the target percentage of the samples based on the following equation, derived from the above equation:
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:
Appendix - 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.
- Plot the average delta OD values on the Y-axis versus the known target level of each standard point on the X-axis.
- Graph the second order polynomial curve* (also see "Example Calculation" below) and obtain the second order polynomial regression equation:
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.
- Calculate the target percentage of the samples based on the following equation, derived from the above equation:
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.
General Product Information
Quality Control: Each lot of SpeedRun™ One-Hour MBD1 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 MBD1 ELISA Kit (Colorimetric) is for research use only and is not intended for diagnostic or therapeutic applications.
Intellectual Property: The SpeedRun™ One-Hour MBD1 ELISA Kit (Colorimetric) and methods of use contain proprietary technologies by EpigenTek.