Polyhistidine-tag ELISA Kit,Absin_specification/price/image

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Product Specification

Usage

Sample Processing and Requirements: Serum: Whole blood samples collected in serum separator tubes should be placed at room temperature for 2 hours or at 4°C overnight. Then, centrifuge at 1000×g for 20 minutes and remove the supernatant. Alternatively, the supernatant may be stored at -20°C or -80°C, but repeated freezing and thawing should be avoided. Plasma: Specimens should be collected using EDTA or heparin as an anticoagulant. Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. The supernatant may be removed for testing, or the supernatant may be stored at -20°C or -80°C, but repeated freezing and thawing should be avoided. Tissue Homogenate: Rinse tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate may affect the measurement results). Weigh and mince the tissue. Mix the minced tissue with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for analysis.
Cell Lysis Buffer: Adherent cells are gently washed with ice-cold PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. The harvested cells are washed three times with ice-cold PBS and resuspended in 150-200µL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, the PBS volume can be reduced appropriately). Disrupt the cells by repeated freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for testing.
Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, and remove the supernatant for testing. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freeze-thaw cycles.
Other biological fluids: Centrifuge at 1000×g for 20 minutes, and remove the supernatant for testing.

Preparation for the test:
1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Preparation of Gradient Standard Working Solution: Add 1 mL of Universal Diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 800 ng/mL). Then dilute to the following concentrations: 800 ng/mL, 400 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, and 0 ng/mL. Serial Dilution Method: Add 500 μL of Universal Diluent to each of seven EP tubes. Pipette 500 μL of the 800 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 400 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube.
3. Prepare Biotin-Antibody Working Solution: 15 minutes before use, centrifuge the concentrated Biotin-Antibody at 1000×g for 1 minute. Dilute 100µL of concentrated Biotin-Antibody to a 1× working concentration with universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Use the same day.
4. Prepare Enzyme Conjugate Working Solution: 15 minutes before use, centrifuge the 100x concentrated enzyme conjugate at 1000×g for 1 minute. Dilute 100µL of concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Use the same day. 5. Prepare 1× Wash Buffer: Dissolve 10 mL of 20× Wash Buffer in 190 mL of distilled water. (Concentrated Wash Buffer removed from the refrigerator may crystallize, which is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing.) Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Sample Addition: Add 50 μL of sample or standard of varying concentrations to the corresponding wells. Add 50 μL of Universal Diluent to the blank wells, followed by 50 μL of Biotin-Antibody Working Solution to each well. Cover with sealant and incubate at 37°C for 1 hour. (Recommendation: Dilute the sample to be tested at least 1x with universal diluent before adding it to the ELISA plate for testing to reduce the influence of matrix effects on the test results. The final calculation of sample concentration should be multiplied by the corresponding dilution factor. It is recommended to set up duplicate wells for all samples and standards.)
3. Wash: Discard the liquid and add 300uL of 1x wash buffer to each well. Let it stand for 1 minute. Shake off the wash buffer and pat dry on absorbent paper. Repeat this process three times (a microplate washer can also be used).
4. Add enzyme conjugate working solution: Add 100uL of enzyme conjugate working solution to each well, cover with a film, and incubate at 37°C for 30 minutes.
5. Wash: Discard the liquid and wash the plate according to step 3 five times.
6. Add substrate: Add 90uL of substrate (TMB) to each well, cover with a film, and incubate at 37°C in the dark for 15 minutes. 7. Add Stop Solution: Remove the ELISA plate and add 50 μL of Stop Solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm. Calculation of Experimental Results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction value. Plot a four-parameter logistic function standard curve on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is above the upper limit of the standard curve, dilute the sample appropriately and re-measure. Multiply the sample concentration by the corresponding dilution factor.

Note: This figure is for reference only. The sample content should be calculated based on the standard curve drawn for the same test standard.

Theory

This kit uses a competitive enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotin-labeled antibodies, and HRP conjugates are sequentially added to microwells pre-coated with a universal polyhistidine-tag antigen. After incubation and washing, the color is developed using the substrate TMB. TMB is converted to blue under the catalysis of peroxidase (HRP) and to yellow under the action of acid. The color intensity is negatively correlated with the universal polyhistidine-tag in the sample. The absorbance (OD value) is measured at a wavelength of 450 nm using a microplate reader to calculate the sample concentration.

Source

All

Synonym

General Polyhistidine-tag ELISA Kit；His Tag ELISA Kit

Detection Type

Competition Law

Composition

Name	9 6 T Configuration	Remarks
Pre-coated 96-well enzyme plate	8 holes×12 strips	None
Standard	2 bottles	Dilution according to the instructions
Universal diluent	2×20mL	None
Concentrated Biotin-Antibody (100×)	60uL	Dilution according to the instructions
Concentrated enzyme conjugate (100×)	120uL	Dilution according to the instructions
20× Wash solution	2×10mL	Dilute according to instructions
Substrate (TMB)	10mL	None
Stop solution	6mL	none
Sealing film	4 sheets	None
Instructions	1 serving	None

Background

polyhistidine tag is an amino acid motif in proteins, typically composed of at least six histidine (His) residues, and is typically located at the N- or C-terminus of the protein. It is also known as a hexahistidine tag, 6xHis tag, or His6 tag. This tag was invented by Roche. Generally speaking, proteins possess a greater or lesser ability to coordinate metal ions on their surfaces, and differences in their affinity can be exploited to separate proteins using chromatography.

General Notes

1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use.
2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation.
3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value.
4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue.
5. Avoid cross-contamination of reagents and specimens to prevent erroneous results.
6. Avoid direct exposure to strong light during storage and incubation.
7. Do not expose any reagents to bleaching agents or the strong fumes emitted by bleaching agents. Any bleaching agent will destroy the biological activity of the reagents in the kit.
8. Do not use expired products, and do not mix components from different product numbers and batches.
9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized.
10. If there is a possibility of disease transmission, all samples should be managed properly and the samples and testing devices should be handled according to the prescribed procedures.

Storage Temp.

Unopened test kit, stored at 2-8°C, has a shelf life of 6 months.

Test Range

12.5-800ng/mL

Applications

Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.