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Rat Pdha1 ELISA Kit

SKU:
RTFI00243
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P26284
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich
Synonyms:
Pdha1, PDHE1-A type I
Reactivity:
Rat
Research Area:
Metabolism
€649
Frequently bought together:

Description

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Rat Pdha1 ELISA Kit

The Rat PDHA1 ELISA Kit is specifically designed for the accurate measurement of pyruvate dehydrogenase E1 component subunit alpha (PDHA1) levels in rat samples including serum, plasma, and tissue homogenates. This kit offers high sensitivity and specificity, ensuring precise and reliable results for various research applications.PDHA1 is a key enzyme in the pyruvate dehydrogenase complex, playing a crucial role in the conversion of pyruvate to acetyl-CoA in the citric acid cycle.

Dysregulation of PDHA1 has been linked to metabolic disorders, neurodegenerative diseases, and cancer, making it a valuable biomarker for studying these conditions and discovering potential therapeutic interventions.Overall, the Rat PDHA1 ELISA Kit provides a convenient and efficient method for researchers to quantify PDHA1 levels in rat samples, facilitating the investigation of metabolic pathways and the development of novel treatment strategies.

Product Name:Rat Pdha1 (Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial) ELISA Kit
Product Code:RTFI00243
Size:96 Assays
Target:Rat Pdha1
Alias:Pdha1, PDHE1-A type I
Reactivity:Rat
Detection Method:Sandwich ELISA, Double Antibody
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Rat Pdha1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Pdha1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)89-10298
EDTA plasma(n=5)91-10597
UFH plasma(n=5)93-104100
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Pdha1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-104%86-99%87-101%
EDTA plasma(n=5)84-99%82-101%84-99%
UFH plasma(n=5)80-93%81-95%81-94%
Intra-Assay:CV <8%
Inter-Assay:CV <10%
Uniprot:P26284
UniProt Protein Function:PDHA1: a mitochondrial matrix enzyme that catalyzes the oxidative decarboxylation of pyruvate, producing acetyl-CoA and CO2. A key enzyme in controlling the balance between lipid and glucose oxidation depending on substrate availability. The pyruvate dehydrogenase (PDH) holoenzyme is a multi-enzyme complex (PDHC) that contains 20-30 copies of pyruvate decarboxylase tetramers (2 alpha:2 beta)(E1), 60 copies of dihydrolipoamide acetyltransferase (E2), six homodimers of dihydrolipoamide dehydrogenase (E3), plus E3 binding proteins. The activity of PDH is tightly regulated by phosphorylation. The phosphorylation of at least one of three specific serine residues in E1 subunit by PDHK inactivates the PDHC, while dephosphorylation by PDP restores its activity. Sites 1, 2, and 3 of PDHA1 are S293, S300, and S232, respectively. Four PDHK isoenzymes have been described, each with different site specificity: all four phosphorylate sites 1 and 2 but at different rates; for site 1 PDHK2 >PDHK4 >PDHK1 >PDHK3; for site 2, PDHK3> PDHK4 > PDHK2 > PDHK1. Only PDHK1 phosphorylates site 3. PDHA1 deficiency is the most common enzyme defect in patients with primary lactic acidosis.
UniProt Protein Details:

Protein type:Mitochondrial; Carbohydrate Metabolism - glycolysis and gluconeogenesis; Carbohydrate Metabolism - pyruvate; Carbohydrate Metabolism - butanoate; Carbohydrate Metabolism - citrate (TCA) cycle; Amino Acid Metabolism - valine, leucine and isoleucine biosynthesis; EC 1.2.4.1; Oxidoreductase

Cellular Component: intracellular membrane-bound organelle; mitochondrial pyruvate dehydrogenase complex; mitochondrion; myelin sheath; nucleus; pyruvate dehydrogenase complex

Molecular Function:pyruvate dehydrogenase (acetyl-transferring) activity; pyruvate dehydrogenase activity

Biological Process: acetyl-CoA biosynthetic process from pyruvate; glucose metabolic process; tricarboxylic acid cycle

NCBI Summary:catalyzes the interconversion of pyruvate and lipoamide to S-acetyldihydrolipoamide and CO2 [RGD, Feb 2006]
UniProt Code:P26284
NCBI GenInfo Identifier:119364627
NCBI Gene ID:29554
NCBI Accession:P26284.2
UniProt Related Accession:P26284
Molecular Weight:
NCBI Full Name:Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial
NCBI Synonym Full Names:pyruvate dehydrogenase E1 alpha 1 subunit
NCBI Official Symbol:Pdha1  
NCBI Protein Information:pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial
UniProt Protein Name:Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial
UniProt Synonym Protein Names:PDHE1-A type I
Protein Family:Pyruvate dehydrogenase E1 component
UniProt Gene Name:Pdha1  
UniProt Entry Name:ODPA_RAT
StepProcedure
1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4. Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37°C for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12. Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol
Serum:

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum and assay promptly or aliquot and store the samples at-80°C. Avoid multiple freeze-thaw cycles.

Plasma: Collect plasma using EDTA or heparin as an anti-coagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid: Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell Culture Supernatant: Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell Lysates: Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenizein 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or-80°C.
Tissue Lysates: Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk: Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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