Customized Rat Glial Cell Line-derived Neurotrophic Factor ELISA Kit GDNF Sandwich Elisa Kit

Customized Rat Glial Cell Line-derived Neurotrophic Factor ELISA Kit GDNF Sandwich Elisa Kit

Cat.No E0351Ra

Standard Curve Range: 0.05ng/ml - 15ng/ml

Sensitivity: 0.02ng/ml

Size: 96 wells

*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.

Precision

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.

CV(%) = SD/mean x 100

Intra-Assay: CV<8%

Inter-Assay: CV<10%

Intended Use

This sandwich kit is for the accurate quantitative detection of Rat Glial Cell Line-derived Neurotrophic Factor (also known as GDNF) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.

Assay Principle

This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Rat GDNF antibody. GDNF present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Rat GDNF Antibody is added and binds to GDNF in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated GDNF antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Rat GDNF. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.

Precautions

• Prior to use, the kit and sample should be warmed naturally to room temperature 30 minutes.
• This instruction must be strictly followed in the experiment.
• Once the desired number of strips has been removed, immediately reseal the bag to protect the remain from deterioration. Cover all reagents when not in use.
• Make sure pipetting order and rate of addition from well-to-well when pipetting reagents.
• Pipette tips and plate sealer in hand should be clean and disposable to avoid cross-contamination.
• Avoid using the reagents from different batches together.
• Substrate solution B is sensitive to light, don’t expose substrate solution B to light for a long time.
• Stop solution contains acid. Please wear eye, hand and skin protection when using this material. Avoid contact of skin or mucous membranes with kit reagent.
• The kit should not be used beyond the expiration date.

Specimen Collection

Serum Allow serum to clot for 10-20 minutes at room temperature. Centrifuge at 2000-3000 RPM for 20 minutes.

Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C within 30 minutes of collection.

Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for approximately 20 minutes. When collecting pleuroperitoneal fluid and cerebrospinal fluid, please follow the procedures above-mentioned.

Cell Culture Supernatant Collect by sterile tubes when examining secrete components. Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect the supernatants carefully. When examining the components within the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge at 2000-3000 RPM for approximately 20 minutes.

Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly and weigh before homogenization. Mince tissues and homogenize them in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20 minutes.

Note

• Sample concentrations should be predicted before being used in the assay. If the sample concentration is not within the range of the standard curve, users must contact us to determine the optimal sample for their particular experiments.
• Samples to be used within 5 days should be stored at 2-8°C. Samples should be aliquoted or must be stored at -20°C within 1 month or -80°C within 6 months. Avoid repeated freeze thaw cycles.
• Samples should be brought to room temperature before starting the assay.
• Centrifuge to collect sample before use.
• Samples containing NaN3 can’t be tested as it inhibits the activity of Horse Radish Peroxidase (HRP).
• Collect the supernatants carefully. When sediments occurred during storage, centrifugation should be performed again.
• Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.

*Sample can't be diluted with this kit. Owing to the the material we use to prepare the kit, the sample matrix interference may falsely depress the specificity and accuracy of the assay.

Assay Procedure

1. Prepare all reagents, standard solutions and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature.

2. Determine the number of strips required for the assay. Insert the strips in the frames for use. The unused strips should be stored at 2-8°C.

3. Add 50μl standard to standard well. Note: Don’t add antibody to standard well because the standard solution contains biotinylated antibody.

4. Add 40μl sample to sample wells and then add 10μl anti-GDNF antibody to sample wells, then add 50μl streptavidin-HRP to sample wells and standard wells (Not blank control well). Mix well. Cover the plate with a sealer. Incubate 60 minutes at 37°C.

5. Remove the sealer and wash the plate 5 times with wash buffer. Soak wells with at least 0.35 ml wash buffer for 30 seconds to 1 minute for each wash. For automated washing, aspirate all wells and wash 5 times with wash buffer, overfilling wells with wash buffer. Blot the plate onto paper towels or other absorbent material.

6. Add 50μl substrate solution A to each well and then add 50μl substrate solution B to each well. Incubate plate covered with a new sealer for 10 minutes at 37°C in the dark.

7. Add 50μl Stop Solution to each well, the blue color will change into yellow immediately.

8. Determine the optical density (OD value) of each well immediately using a microplate reader set to 450 nm within 10 minuets after adding the stop solution.

Calculation of Result

Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.

Referances

"Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis."
Michos O., Goncalves A., Lopez-Rios J., Tiecke E., Naillat F., Beier K., Galli A., Vainio S., Zeller R.
Development 134:2397-2405(2007)

Shanghai Korain Biotech Co. was built in 2010. As an creator in reagents and tools for life science, Korainbio provide researchers with tools and scientific support including 30,000 antibodies, 1000+ proteins and 5000 ELISA kits. We aim to be a leading provider with world-class level for the researchers all over the world. Our products line covers a set of research areas including Immunology, Neuroscience, Cancer, Kinases, Phosphatases and Cell Biology.

Bioassay Technology Laboratory (BT Lab) as a leading brand of Korainbio focused on helping researchers to optimize life science work and serves as a provider of test and developing services, including elisa tests, WB test and antigen development. Founding in 2010, our strategic focus has been on the development of enabling technologies in the research, development, manufacture and marketing of innovative immuno products and services based on molecular technologies.

Korain’s reagents are supported by superior processional team and a quality management system that is certified for CE and ISO. Our product development program for use in a variety of applications including:

ELISA and immunohistochemistry

• Immunoprecipitation
• Immunohistochemistry
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• Immunohistochemistry
• Quantitative Multiplexing

About BT Lab

As a leading brand of Korainbio, Bioassay Technology Laboratory (BT Lab) offers a variety of cost-effective ELISA kits and sets to measure cytokines, chemokines, and soluble biomarkers consistently and reliably. Sandwich kit and Competitive that provide the core reagents our selection of ELISA products meets the demand of ELISA beginners to experts alike at a very economical price.

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ELISA Kit Categories

Sandwich Kit
Sandwich kits are fully validated and ready-to-use, containing 96- well strip plates pre-coated with capture antibody to detect sample antigen.

Competitive Kit
In a competitive ELISA assay, sample antigen and labeled antigen compete for capture antibody binding. The more target protein there is in the sample, the less labeled antigen will be captured and the weaker the signal.

Qualitative Kit
Qualitative results provide a simple positive or negative result for a sample. In quantitative ELISA, the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule. It is suitable for detecting small antigens.

Advantages of our Sandwich kit

Without Diluting Sample

• Save assay time
• Wide detection range
• Reducing non-specific interactions between the sample matrix proteins

Streptavidin-biotin system

• Significant non-specific binding can be prevented by strepvidin
• Reducing interference from residual biotin in sample
• A non-covalent interaction with high affinity

Washing Step

• Only 5 times for one step

Pre-Coated plate

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