Epitope tags provide a convenient way to isolate interacting proteins without the need for specific antibodies to each new protein. His-tag is a short amino acid sequence consists of histidine (His) residues in recombinant proteins.

ABSBio™ His-tag Protein ELISA Kit provide a convenient, ready-to-use, high-throughput platform for rapid capture and detection of His-tagged proteins in different samples. The High Sensitivity plate is coated with the His-tagged protein and pre-blocked to provide timesaving for high-throughput users. Series dilutions of His-protein standards (28kD) and samples are added to each test well, then, anti-His tag mAb is added to the plate. His-tagged protein in solution and pre-coated His-tagged protein compete to bind the antibody. After washing steps, HRP-conjugated secondary antibody is added to each well to react with anti-His tag mAb. Finally, TMB substrates is added, incubated for detection, and a blue color is developed. Reaction is stopped and color turns to yellow when Stopping Solution is added. The yellow color intensity proportionally correlates to the concentration of the His-tagged protein standard and His-tagged protein in samples.

ABSbio’s kit is a competitive assay that generates a reverse curve, with the lowest OD values obtained from high His concentrations, and allows you to detect and quantify His-tagged protein samples simply and reliably by comparing your unknown samples to a known recombinant standard. The kit can be used to detect N-terminal and C-terminal His-tagged proteins. This kit contains all reagents required for His-tag protein detection, suitable for most samples, such as bacterial, yeast, mammalian cell lysates and cell culture supernatants, and the test completed within 1.5 hours. The kit can detect as little as 1 ng/mL His-tagged protein in samples.

试剂盒组分

储存和处理: Shipping on ice. Store Standard & Detection Ab at -20°C, other components at 2-8°C. Shelf life: 3 months after receipt. Warm Reagents to room temperature before use.

应用:Measurement of His fusion protein in serum, plasma, culture media and cell lysate.

反应物种:Human, Mouse, Rat

样品类型:serum, plasma, cell and tissue culture supernatant, and homogenate cell and tissue samples

检测方法:Colorimetric Sandwish ELISA assay (OD450 nm)

检测时间:1.5 hrs

储存:-20°C & 2-8°C

保质期:3 months after receipt.

运输:Icepacks

所需其他材料:
Multi-channel pipette for washing

Cell culture incubator

Orbital shaker

micro plate reader

试剂准备

1. Wash Solution: 10x dilute Wash Solution with dH2O to prepare 1x Wash Solution.

2. His-tagged Protein Standard (1 vial): The undiluted standard can be stored at -20° C for up to 3 months if not used immediately. Spin to bring down the material prior to open the tube. Add 10 μl of the standard to 500 μl of Assay Diluent to make the high standard concentration of 1000 ng/ml. A seven point standard curve is generated using 3-fold serial dilutions in Assay Diluent, vortex briefly for each of dilution step. Store the rest of the standard at -20° C.

3. Anti-His Detection Ab: Immiediately before use, add 30 µl of the anti-his antibody into 6 ml of Assay Diluent for one plate(for partial plate assay, adjust the volumes accordingly, store the rest of the abtibody at -20°C).

4. HRP-conjugated Ab:Immiediately before use, add 55 µl of the antibody into 11 ml of Assay Diluent for one plate(for partial plate assay, adjust the volumes accordingly).

程序指南

1. Prepare sample dilutions (series 10 fold dilution) in a clean 96‐well plate with 1x PBS.

2. Set standard wells, testing sample and blank wells on the assay plate/strip as above. Transfer diluted standard 50 μl to standard wells, diluted sample 50 μl to sample wells, 50 μl Assay diluent only to blank wells. Assay should be run in duplicate. Incubate at RT for 5 mins on a micro-plate shaker at 600-700 rpm, then add 50 μl of the prepared Detection Ab to all wells.

3. Cover the plate with plate sealer and incubate the plate at room temperature for 30 mins, shaking the plate at 600-700 rpm on a micro-plate shaker.

4. Decant as much liquid as possible, fill the wells with 250 μl wash solution, shaking the plate at 600-700 rpm on a micro-plate shaker for 5 mins, decant the wash solution and remove residual liquid with absorbent paper. Repeat wash THREE times.

5. Add 100 µl of the prepared HRP-conjugated Ab solution to all wells.

6. Cover the plate with plate sealer and incubate at room temperature for 30 mins, shaking the plate at 600-700 rpm on a micro-plate shaker.

7. Wash the plate FIVE times as described in Step#4.

8. Add 100 µl of TMB Solution to each well and incubate at room temperature for 10~20 minutes, or keep close monitoring on the developing process until desired developing blue color observed.

9. Add 100 µl of Stop Solution to each well to stop the reaction (the blue color change to yellow).

10. Read absorbance of the plate on a microplate reader at 450 nm within 15 min.

11. Average the duplicate readings for each standard and samples, subtract the average zero (blank) standard optical density. Construct standard curve (plotting the mean OD450 for each standard on the X‐axis against concentration on the Y‐axis, draw a best‐fit curve through the points) and calculate linear regression equation, then use corrected sample OD values and regression equation to calculate the corresponding sample concentration. It should be remembered that the sample has been diluted and its actual concentration should be justified by dilution factor (the measurement and calculation can be accomplished by software like SoftMax).

12. If molecular weight of sample differs from His-tagged standard (MW 28kD) apply the following equation to the reading concentration to obtain the actual concentration =[MW Sample]/[MW protein standard] x Sample reading (ng/mL)