Porcine ABAT ELISA KIT

Porcine ABAT ELISA KIT

Packing  96Tests

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ ENTIRE PROCEDURE!

Gene Name  ABAT

Protein Name  4-aminobutyrate aminotransferase

Alternative Name

ABAT;GABA-AT; GABAT; NPD009; 4-aminobutyrate aminotransferase

Porcine ABAT ELISA Kit Detect Range  0.15-10ng/mL

Intended use  

Porcine ABAT ELISA KIT allows for the in vitro quantitative determination of ABAT concentrations in serum, plasma, tissue homogenates, cell culture supernates or other biological fluids.

Test principle  

The microtiter plate provided in Porcine ABAT ELISA KIT has been pre-coated with an ABAT antibody specific to ABAT. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for ABAT and then avidin conjugated to Horseradish Peroxidase (HRP) is added  to  each  microplate  well  and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain ABAT, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of ABAT in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Sample collection and storage        

Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20鈩?/span>

or -80鈩?/span> .

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples  for 15 minutes at 1000 × g at 2鈩?/span>- 8鈩?/span>  within 30 minutes of collection. Store samples at -20鈩?/span>  or -80鈩?/span> .

Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with ice-cold 1×PBS to remove excess blood, homogenized in ice-cold 1×PBS and stored overnight at ≤-20鈩?/span> . In most cases, 10% homogenate (eg.1g of tissue in 10mL of ice-cold 1×PBS) is recommended. After two freeze-thaw cycles were performed  to  break the cell membranes, the homogenates were  centrifuged

for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤-20鈩?/span>  .

Cell culture supernates and Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20鈩?/span> or -80鈩?/span> .

Fresh  samples are first choice. If not, avoid freeze-thaw of samples.

Reagent preparation      

Standard - Please refer to the Data Sheet inserting in the ELISA kit.

Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.

Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have  completely dissolved.  Dilute 30mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer.

Assay procedure     

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37鈩?/span> directly). All the reagents should be mixed thoroughly by gently  swirling  before  pipetting.  Avoid foaming. Keep appropriate  numbers

of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20鈩?/span> until the kits expiry date. Prepare  all  reagents,  working  standards  and  samples  as  directed  in the

previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular  experiments.

1.     Add 100 μL of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37鈩?/span>.

2.     Remove  the  liquid  of  each  well,  don’t  wash.  Add  100μL  of  Detection

Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37鈩?/span>. Detection Reagent A working solution may appear  cloudy.  Warm  to  room  temperature and mix gently until solution

appears uniform.

3.     Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

4.     Add 100μL of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hour at 37鈩?/span>.

5.     Repeat the aspiration/wash process for 5 times as conducted in step 3.

6.     Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 15-30 minutes at 37鈩?/span>. Protect from light.

7.     Add  50μL of  Stop  Solution  to each well. If color change does not appear

uniform, gently tap the plate to ensure thorough mixing.

8.     Determi