Mouse MIP3b 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. Prepare a 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 1000 pg/mL). Then dilute to the following concentrations: 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.625 pg/mL, and 0 pg/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 1000 pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500 pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent). Prepare the solution immediately before use. 4. Preparation of Enzyme Conjugate Working Solution: 15 minutes before use, centrifuge 100 μL of concentrated enzyme conjugate at 1000 μg for 1 minute. Dilute 100 μL of concentrated HRP enzyme conjugate with universal diluent to a 1 μL working concentration (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare freshly for use. 5. Preparation of 1 μL Wash Solution: Dissolve 10 mL of 20 μL Wash Solution in 190 mL of distilled water. (Crystallization may occur in the concentrated wash solution after removal from the refrigerator. This is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparation.) Procedure: 1. After equilibration at room temperature for 10 minutes, remove the desired strips from the aluminum foil bag. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Sample Addition: Add 100 μL of sample or standard of varying concentrations to the appropriate wells. Add 100 μL of universal diluent to the blank wells. Cover with film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the test samples at least 1-fold with universal diluent before adding them to the plate. This will minimize matrix effects on the test results. When calculating sample concentrations, multiply by the dilution factor. It is recommended to run replicates for all test samples and standards.) 3. Biotinylated Antibody Addition: Remove the plate, discard the liquid, and do not wash. Add 100 μL of biotinylated antibody working solution directly to each well. Cover with film and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x wash buffer to each well. Let 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). 5. Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with a film sealer, and incubate at 37°C for 30 minutes. 6. Wash: Discard the liquid and wash the plate five times according to the procedure in step 4. 7. Add substrate: Add 90 μL of substrate (TMB) to each well, cover with a film sealer, and incubate at 37°C in the dark for 15 minutes. 8. 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 450 nm. Calculating Experimental Results:
Interpreting Results: 1. Calculate the average OD value of the replicate standard and sample wells and subtract the blank well OD value as a correction value. Plot a standard curve for the four-parameter logistic function 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 higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor when calculating the sample concentration.

Note: This graph is for reference only. Sample content should be calculated using the standard curve plotted for each experimental data.

Theory

This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a capture antibody against Macrophage Inflammatory Protein 3 Beta (MIP3b). After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of Macrophage Inflammatory Protein 3 Beta (MIP3b) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration.

Source

Mouse

Synonym

Mouse Macrophage Inflammatory Protein 3 Beta ELISA Kit, Mouse CCL19 ELISA Kit

Detection Type

Double antibody sandwich method

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 biotinylated detection antibody (100×)	120uL	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

Macrophage inflammatory protein 3β (MIP-3β/CCL19) is a protein encoded by the CCL19 gene. It is a small cytokine belonging to the CC chemokine family. This gene is one of several CC cytokine genes clustered on the p arm of chromosome 9. The cytokine encoded by this gene may play a role in normal lymphocyte recirculation and homing. It also plays an important role in T cell trafficking in the thymus and the migration of T and B cells to secondary lymphoid organs. This chemokine exerts its effects on target cells by binding to the chemokine receptor CCR7. It attracts certain cells of the immune system, including dendritic cells, antigen-engaged B cells, and CCR7+ central memory T cells.

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.

Store at 2-8°C. Valid for 6 months.

Test Range

15.62-1000 pg/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.