PCK1 ELISA Kit

Catalog No. ABIN456132

Product Details PCK1 ELISA Kit

Target: PCK1 (phosphoenolpyruvate Carboxykinase 1 (Soluble) (PCK1))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.312-20 ng/mL

Minimum Detection Limit: 0.312 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the in vitro quantitative determination of human PCK concentrations in tissue homogenates and other biological fluids.

Sample Type: Tissue Homogenate

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural human PCK.

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Sensitivity: < 0.078 ng/mL
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Characteristics: Homo sapiens,Human,Phosphoenolpyruvate carboxykinase, cytosolic [GTP],PEPCK-C,Phosphoenolpyruvate carboxylase,PCK1,PEPCK1,4.1.1.32

Components: Reagent (Quantity): Assay plate (1×20ml), Standard (2), Sample Diluent (1×20ml), Assay Diluent A (1×10ml), Assay Diluent B (1×10ml), Detection Reagent A (1×120 μl), Detection Reagent B (1×120 μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), Instruction (1)

Material not included: Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: The microtiter plate provided in this kit has been pre-coated with an antibody specific to PCK. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for PCK and 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 PCK, 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 PCK in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reagent Preparation:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 20 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (20 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). ng/ml 20 10 5 2.5 1.25 0.625 0.312 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.

Sample Collection: Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20 C . 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 C . Other biological fluids - Remove particulates by centrifugation and assay immediately or 3 aliquot and store samples at -20 C or -80 C . Avoid repeated freeze-thaw cycles. Note: Tissue homogenates and cell culture supernatant samples to be used within 7 days may be stored at 2-8 C , otherwise samples must stored at -20 C ( ≤ 1 months) or -80 C ( ≤ 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

Assay Procedure:

4 Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 C 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 4 C 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 μ l of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 C .
2. Remove the liquid of each well, don ’ t wash.
3. Add 100 μ l of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 C . Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
4. 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 μ l) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to 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.
5. Add 100 μ l of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 C .
6. Repeat the aspiration/wash as in step
4. 7. Add 90 μ l of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37 C . Protect from light.
8. Add 50 μ l of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
Important Note:
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10 μ l for once pipetting.
3. To ensure accurate results, proper adhesion of plate sealers during incubation steps 5 is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.

Calculation of Results:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the PCK concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for PCK1

Target: PCK1 (phosphoenolpyruvate Carboxykinase 1 (Soluble) (PCK1))

Alternative Name: PCK1 (PCK1 Products)

Synonyms: PEPCK-C ELISA Kit, PEPCK1 ELISA Kit, PEPCKC ELISA Kit, PEPCK ELISA Kit, PEPCK-M ELISA Kit, PEPCK2 ELISA Kit, 1810010O14Rik ELISA Kit, 9130022B02Rik ELISA Kit, cb856 ELISA Kit, cb924 ELISA Kit, fj93h11 ELISA Kit, zgc:63869 ELISA Kit, wu:fc51c05 ELISA Kit, wu:fj93h11 ELISA Kit, pepck-c ELISA Kit, pepck1 ELISA Kit, pepckc ELISA Kit, PCK1 ELISA Kit, PPCK1 ELISA Kit, AI265463 ELISA Kit, Pck-1 ELISA Kit, GTP ELISA Kit, PCK ELISA Kit, Pepck ELISA Kit, RATPEPCK ELISA Kit, Ppc1C ELISA Kit, PHOSPHOENOLPYRUVATE CARBOXYKINASE ELISA Kit, T28I19.150 ELISA Kit, T28I19_150 ELISA Kit, phosphoenolpyruvate carboxykinase 1 ELISA Kit, 143299_at ELISA Kit, CG10924 ELISA Kit, CG17725 ELISA Kit, Dmel\\CG17725 ELISA Kit, Dromel_CG17725_FBtr0086701_pepck_mORF ELISA Kit, dPEPCK ELISA Kit, pepck ELISA Kit, PEPC ELISA Kit, PEPCase ELISA Kit, ppc ELISA Kit, phosphoenolpyruvate carboxykinase 1 ELISA Kit, phosphoenolpyruvate carboxykinase 2, mitochondrial ELISA Kit, phosphoenolpyruvate carboxykinase 2 (mitochondrial) ELISA Kit, phosphoenolpyruvate carboxykinase 1 (soluble) ELISA Kit, phosphoenolpyruvate carboxykinase 1 S homeolog ELISA Kit, phosphoenolpyruvate carboxykinase, cytosolic [GTP] ELISA Kit, phosphoenolpyruvate carboxykinase 1, cytosolic ELISA Kit, phosphoenolpyruvate carboxylase 7 ELISA Kit, Phosphoenolpyruvate carboxykinase ELISA Kit, phosphoenolpyruvate carboxylase ELISA Kit, PCK1 ELISA Kit, PCK2 ELISA Kit, Pck2 ELISA Kit, pck1 ELISA Kit, pck1.S ELISA Kit, LOC100634531 ELISA Kit, Pck1 ELISA Kit, pep7 ELISA Kit, Pepck ELISA Kit, LOC107777405 ELISA Kit

Background: Phosphoenolpyruvate carboxykinase (or PEPCK) is an enzyme used in the natural process of gluconeogenesis. It converts oxaloacetate into phosphoenolpyruvate and carbon dioxide. It is found in two forms, cytosolic and mitochondrial. Whereas most reactions of gluconeogenesis can use the glycolysis enzymes in the opposite direction, the pyruvate kinase enzyme is irreversible. Therefore, the enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase are used to provide an alternate path for effectively reversing its actions. PEPCK gene transcription (genetics) occurs in many species, and the amino acid sequence of PEPCK is distinct for each species. For example, its structure and its specificity differ in humans, Escherichia coli (E. coli), and the parasiteTrypanosoma cruzi. It has been shown that PEPCK catalyzes the reversible rate-controlling step of gluconeogenesis, the process whereby glucose is synthesized. The enzyme has therefore been thought to be essential in glucose homeostasis, as evidenced by laboratory mice that contracted diabetes mellitus type 2 as a result of the overexpression of PEPCK. A recent study suggests that the role that PEPCK plays in gluconeogenesis may be mediated by the citric acid cycle, the activity of which was found to be directly related to PEPCK abundance. PEPCK levels alone were not found to be highly correlated with gluconeogenesis in the mouse liver, as previous studies have suggested. Therefore, the role of PEPCK in gluconeogenesis may be more complex and involve more factors than was previously believed. PEPCK of Mycobacterium tuberculosis has been shown to trigger the immune system in mice by increasing cytokine activity. As a result, it has been found that PEPCK may be an 2 appropriate ingredient in the development of an effective subunit vaccination for tuberculosis.

Pathways: Positive Regulation of Peptide Hormone Secretion, Carbohydrate Homeostasis