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Human JAM-A ( PAM-1 / CD321 ) ELISA Kit
 
 
 
 
 
 
 
Order or Inquire for Human JAM-A / PAM-1 / CD321 ELISA Kit 
 
 
 
 
 
 
High affinity Human JAM-A / PAM-1 / CD321 ELISA Kit
 
 
 
 
 
 
Detection limit - 4.88 pg/ml
 
 
 
 
 
 
Affordable price and 30%-80% cost saving for Bulk order
 
 
 
 
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Materials
 
Protocol
 
Trouble Shooting
 
Image & Flash
 
 
 
 
1. Materials provided
 
Capture Antibody - 0.5 mg/mL of mouse anti-JAM-A monoclonal antibody. Dilute to a working concentration of 2 μg/mL in CBS before coating.
 
Detection Antibody - Each vial contains 120 μg biotinylated rabbit anti-JAM-A polyclonal antibody. Reconstitute with sterile 1 mL distilled water. Dilute to a working concentration of 1 μg/mL in detection antibody dilution buffer before use.
 
Standard - Each vial contains 18 ng of recombinant JAM-A. Reconstitute standard powder with sterile distilled water. A seven-point standard curve using 2-fold serial dilutions in sample dilution buffer, and a high standard of 312.5 pg/mL is recommended.
 
Streptavidin-HRP - 50 μL of streptavidin conjugated to horseradish-peroxidase. 1:2000  Dilution in detection antibody dilution buffer before use.
 
2. Sensitivity
 
The minimum detectable dose of human JAM-A ( PAM-1 / CD321 ) was determined to be approximately 4.88 pg/ml. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
 
3. Principle of the product
 
The human JAM-A ( PAM-1 / CD321 ) ELISA kit is for the quantitative determination of human JAM-A.
 
This ELISA kit contains the basic components required for the development of sandwich ELISAs. Each kit contains sufficient materials to run ELISAs on five 96-well plates.
 
The Sino Biological ELISA kit is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes a monoclonal antibody specific for JAM-A ( PAM-1 / CD321 ) coated on a 96-well plate. Standards and samples are added to the wells, and any JAM-A ( PAM-1 / CD321 ) present binds to the immobilized antibody. The wells are washed and a biotinylated rabbit anti- JAM-A polyclonal antibody is then added, producing an antibody-antigen-antibody “sandwich”. To produces color in proportion to the amount of JAM-A ( PAM-1 / CD321 ) present in the sample strepavidin-HRP and TMB substrate solution are loaded. The absorbances of the microwell are read at 450 nm.
 
STORAGE - Keep streptavidin-HRP at 4℃ and protect it from prolonged exposure to light. Aliquot all other reagents and store at -20℃ to -70℃ in a manual defrost freezer.
 
 
 
Plate Preparation
 
 
Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4℃.
 
Aspirate each well and wash with at least 300μl wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is 3. essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
 
Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
 
Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
 
 
 
 
Assay Procedure
 
 
Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at room temperature.
 
Repeat the aspiration/wash as in step 2 of plate preparation.
 
Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1 hour at room temperature.
 
Repeat the aspiration/wash as in step 2 of plate preparation.
 
Add 100 μL of Streptavidin-HRP to each well. Incubate for 1 hour at room temperature.
 
Repeat the aspiration/wash as in step 2 of plate preparation.
 
Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature ( if substrate solution is not as requested, the incubation time should be optimized ). Avoid placing the plate in direct light.
 
Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
 
Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
 
 
 
 
Calculation of results
 
 
Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each.
 
Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph.
 
To determine the concentration of the unknowns, find the unknowns’ mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
 
Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Problems 
 
 
 
 Possible Sources 
 
 
 
 Solutions 
 
 
 
 
 
 No signal 
 
 
 
Incorrect or no Detection Antibody was added
 
 
 
Add appropriate Detection Antibody and continue
 
 
 
 
 
Substrate solution was not added
 
 
 
Add substrate solution and continue
 
 
 
 
 
Incorrect storage condition
 
 
 
Check if the kit is stored at recommended condition and used before expiration date
 
 
 
 
 
 
 
 
 
 
 
 
 
Poor Standard Curve
 
 
 
Standard was incompletely reconstituted or was inappropriately stored
 
 
 
Aliquot reconstituted standard and store at  -70 ℃
 
 
 
 
 
Imprecise / inaccurate pipetting
 
 
 
Check / calibrate pipettes
 
 
 
 
 
Incubations done at inappropriate temperature, timing or agitation
 
 
 
Follow the general ELISA protocol
 
 
 
 
 
Background wells were contaminated
 
 
 
Avoid cross contamination by using the sealer appropriately
 
 
 
 
 
 
 
 
 
 
 
 
 
Poor detection value 
 
 
 
The concentration of antigen in samples was too low
 
 
 
Enriching samples to increase the concentration of antigen
 
 
 
 
 
Samples were ineffective
 
 
 
Check if the samples are stored at cold environment. Detect samples in timely manner
 
 
 
 
 
 
 
 
 
 
 
 
 
High Background 
 
 
Insufficient washes
 
 
Use multichannel pipettes without touching the reagents on the plate
 
 
 
 
 
Increase cycles of washes and soaking time between washes
 
 
 
 
 
TMB Substrate Solution was contaminated
 
 
 
TMB Substrate Solution should be clear and colorless prior to addition to wells
 
 
 
 
 
Materials were contaminated
 
 
 
Use clean plates, tubes and pipettes tips
 
 
 
 
 
 
 
 
 
 
 
 
 
Non-specificity 
 
 
 
Samples were contaminated
 
 
 
Avoid cross contamination of samples
 
 
 
 
 
The concentration of samples was too high
 
 
 
Try higher dilution rate of samples
 
 
 
 
 
 
 
Human JAM-A / PAM-1 / CD321 ELISA Kit Image
 
 
This standard curve is only for demonstration purposes. A standard curve should be generated for each assay.
 
 
 
Human JAM-A / PAM-1 / CD321 ELISA Kit Flash
  
This flash is a vivid summary of the assay procedure.
 
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Human JAM-A ( PAM-1 / CD321 ) ELISA Kit Related Products & Topics
 
 
相关研究领域:
 
Cancer>>Angiogenesis>>Adhesion Molecules in Angiogenesis>>JAM-A/F11R/CD321
 
Immunology>>Adhesion Molecule>> Cell Adhesion Molecule (IgSF CAM) >>JAM-A/F11R/CD321
 
Immunology>>Cluster of Differentiation>>Other>>JAM-A/F11R/CD321
 
蛋白:
 
 
 
 
Molecule
 
Species
 
Description //For Detailed Info.------CLICK!
 
Cat No
 
Size/Price
 
 
 
JAM-A/F11R/CD321
 
Human
 
JAM-A/F11R/Fc Protein, Recombinant
 
10198-H02H
 
100µg($290); Order
 
 
 
JAM-A/F11R/CD321
 
Human
 
JAM-A/F11R Protein, Recombinant
 
10198-H08H
 
100µg($290); Order
 
 
 
 
抗体:
 
 
 
 
Molecule
 
Application
 
Description //For Detailed Info.------CLICK!
 
Cat No
 
Size/Price
 
 
 
Human 
 JAM-A/F11R/CD321
 
ELISA
 
Mouse Monoclonal Antibody
 
 10198-MM02 
 
 200µg($95) 500µg($195)  Order
 
 
 
Human 
 JAM-A/F11R/CD321
 
WB
 
Mouse Monoclonal Antibody
 
 10198-MM03
 
 200µg($95) 500µg($195)  Order
 
 
 
Human 
 JAM-A/F11R/CD321
 
ELISA
 
Rabbit Polyclonal Antibody
 
10198-RP01
 
 500µg($150) 1mg($240)  Order
 
 
 
Human 
 JAM-A/F11R/CD321
 
WB, ELISA
 
Rabbit Polyclonal Antibody (Antigen Affinity Purified)
 
10198-RP04
 
100µg($250); Order
 
 
 
 
 
Background
 
 
As a typeⅠtransmembrane receptor belonging to the immunoglobulin superfamily, the human JAM-A, also known as platelet adhesion molecule 1 (PAM-1) and platelet F11 receptor, is the firstly identified member of the Junctional Adhesion Molecule (JAM) family comprising at least three members. JAM-A is specifically localized in tight junctions of epithelial and endothelial cells and is involved in the regulation of junctional integrity and permeability by serving as a physical barrier in a homophilic manner. JAM-A is also expressed on the surface of hematopoeitic cells, such as platelets and leukocytes, and acts as a ligand for integrin LFA-1 or a platelet receptor, consequently plays key roles in a variety of cellular processes, including platelet aggregation, leukocyte transmigration, and angiogenesis. In addition, JAM-A has also been implicated in the attachment of reovirus that causes diarrhea in human.
 
 
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