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Porcine HSPG ELISA Kit

SKU:
PRES00044
€649
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Description

Technical Manual

Porcine HSPG ELISA Kit

How does our Porcine HSPG ELISA Kit work?

This kit uses the Sandwich ELISA principle. The ELISA plate provided in this kit has been precoated with an antibody specific to Heparan Sulfate Proteoglycan. Standards or samples are added to the ELISA plate wells and combined with the pre-coated antibodies. Then a biotinylated detection antibody specific for Heparan Sulfate Proteoglycan and AvidinHorseradish Peroxidase (HRP) conjugate are added successively to each plate well and incubated. Free components are washed away, and the substrate solution is then added to each well.

Only wells that contain Heparan Sulfate Proteoglycan, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Heparan Sulfate Proteoglycan. The concentration of Heparan Sulfate Proteoglycan can be calculated in the samples by comparing the OD of the samples to the standard curve.

SARS-CoV-2 Spike Protein S1 RBD ELISA Kit Contents

Component 24T 96T Storage

Micro ELISA Plate (Dismountable)

8 wells x 3 strips

8 wells x 12 strips

-20°C, 6 months

Reference Standard

1 vial

2 vials

-20„ƒ, 6 months

Concentrated Biotinylated Detection Ab(100×)

1 vial, 60 μL

1 vial, 120 μL

-20„ƒ, 6 months

Concentrated HRP Conjugate (100×)

1 vial, 60 μL

1 vial, 120 μL

-20„ƒ, 6 months (Protect from light)

Reference Standard & Sample Diluent

1 vial, 20 mL

1 vial, 20 mL

4„ƒ, 6 months

Biotinylated Detection Ab Diluent

1 vial, 14 mL

1 vial, 14 mL

4„ƒ, 6 months

HRP Conjugate Diluent

1 vial, 14 mL

1 vial, 14 mL

4„ƒ, 6 months

Concentrated Wash Buffer(25×)

1 vial, 30 mL

1 vial, 30 mL

4„ƒ, 6 months

Substrate Reagent

1 vial, 10 mL

1 vial, 10 mL

4„ƒ, 6 months (Protect from Light)

Stop Solution

1 vial, 10 mL

1 vial, 10 mL

4„ƒ

Plate Sealer

5 pieces

5 pieces

Other materials required

  1. Microplate Reader with 450 nm wavelength filter or dual-wavelength (450/630 nm)
  2. High-precision transfer pettor, EP tubes and disposable pipette tips
  3. Incubator capable of maintaining 37„ƒ
  4. Deionized or distilled water
  5. Absorbent paper

Sample collection and preparation

Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 15 min at 1000×g at 2~8°C. Collect the supernatant to carry out the assay. Blood collection tubes should be disposable and be endotoxin-free.

Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 min at 1000×g at 2~8°C within 30 min of collection. Collect the supernatant to carry out the assay. Hemolysed samples are not suitable for ELISA assay.

Cell lysates: For adherent cells, gently wash the cells with pre-cooled PBS and dissociate the cells using trypsin. Collect the cell suspension in a tube and centrifuge for 5 min at 1000×g. Discard the medium and wash the cells 3 times with precooled PBS. For each 1×106 cells, add 150-250µL of pre-cooled PBS to keep the cells suspended. Optimal cell concentration is 1 million/ml. To release cellular components, dilute the cell pellet in PBS and use 3-4 freezethaw cycles in liquid Nitrogen (commercial lyses buffers can be used according to manufacturer’s instructions). Centrifuge at 4°C for 20 mins at 2000-3000 rpm to pellet debris and remove clear supernatant to clean microcentrifuge tube for ELISA or storage.

Tissue homogenates: It is recommended to get detailed references from the literature before analyzing different tissue types. For general information, hemolysed blood may affect the results, so the tissues should be minced into small pieces and rinsed in ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (tissue weight (g): PBS (mL) volume=1:9) with a glass homogenizer on ice. To further break down the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5 min at 5000×g to get the supernatant.

Cell culture supernatant or other biological fluids: Centrifuge samples for 20 min at 1000×g at 2~ 8°C. Collect the supernatant to carry out the assay.

Note for samples:

  1. Samples should be assayed within 7 days when stored at 4°C. Otherwise samples must be aliquoted and stored at -20°C (≤1 month) or -80°C (≤3 months). Avoid repeated freeze-thaw cycles.
  2. Determine the protein concentration before assaying. If the sample concentration is not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
  3. If the sample type is not included in the manual, a preliminary experiment is suggested to verify the validity.
  4. If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibility of causing a deviation.
  5. Some recombinant proteins may not be detected due to a mismatch with the coated antibody or detection antibody.

Reagent Preparation

  1. Bring all reagents to room temperature (18~25°C) before use. Follow the microplate reader manual for set-up and preheat it for 15 min before OD measurement.
  2. Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer with 720 mL of deionized or distilled water to prepare 750 mL of Wash Buffer. Note: if crystals have formed in the concentrate, warm it in a 40°C water bath and mix it gently until the crystals have completely dissolved.
  3. Standard working solution: Centrifuge the standard at 10,000×g for 1 min. Add 1 mL of Reference Standard & Sample Diluent, let it stand for 10 min and invert it gently several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution produces a working solution of 20 ng/mL . Then make serial dilutions as needed. The recommended dilution gradient is as follows: 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0 ng/mL.
    Dilution method: Take 7 EP tubes, add 500µL of Reference Standard & Sample Diluent to each tube. Pipette 500µL of the 20 ng/mL working solution to the first tube and mix up to produce a 10 ng/mL working solution. Pipette 500µL of the solution from the former tube into the latter one according to these steps. The illustration below is for reference.
  4. Biotinylated Detection Ab working solution: Calculate the required amount before the experiment (100µL/well). In preparation, slightly more than calculated should be prepared. Centrifuge the stock tube before use, dilute the 100× Concentrated Biotinylated Detection Ab to 1×working solution with Biotinylated Detection Ab Diluent.
  5. Concentrated HRP Conjugate working solution: Calculate the required amount before the experiment (100µL/well). In preparation, slightly more than calculated should be prepared. Dilute the 100× Concentrated HRP Conjugate to 1× working solution with Concentrated HRP Conjugate Diluent.

Porcine HSPG ELISA Kit Assay Procedure

  1. Set standard, test sample and control (zero) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Note: add all solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensure solutions do not foam when adding to the wells.
  2. Aliquot 100µl of standard solutions into the standard wells.
  3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
  4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and other biological fluids) into test sample wells.
  5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
  6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of Biotinylated Detection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
  7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each well and incubate for 1-2 minutes at room temperature. Aspirate the solution from each well and clap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplate washer can be used in this step and other wash steps.
  8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal and incubate for 30 min at 37°C.
  9. Aspirate or decant the solution from each well. Repeat the wash process for five times as conducted in step 7.
  10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate for approximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can be shortened or extended according to the actual color change, but not by more than 30min.
  11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done in the same order as the substrate solution.
  12. Determine the optical density (OD value) of each well immediately with a microplate reader set at 450 nm.