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Rat Prolyl 4-hydroxylase subunit alpha-1 / P4HA1 ELISA Kit

SKU:
RTFI00299
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P54001
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich
Synonyms:
P4ha1, PHalphaI
Reactivity:
Rat
€649
Frequently bought together:

Description

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Rat Prolyl 4-hydroxylase subunit alpha-1/P4HA1 ELISA Kit

The Rat Prolyl 4-Hydroxylase Subunit Alpha-1 (P4HA1) ELISA Kit is a powerful tool designed for the precise measurement of P4HA1 levels in rat serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for a variety of research applications.P4HA1 plays a critical role in collagen synthesis, specifically in the hydroxylation of proline residues essential for collagen stability. Dysregulation of P4HA1 has been implicated in various diseases such as fibrosis, cancer, and connective tissue disorders, highlighting its importance as a potential therapeutic target and biomarker.

By utilizing the Rat P4HA1 ELISA Kit, researchers can gain valuable insights into the role of P4HA1 in disease pathogenesis and progression, paving the way for the development of novel treatment strategies. Trust in the reliability and precision of this ELISA kit for your research needs.

Product Name:Rat P4ha1 (Prolyl 4-hydroxylase subunit alpha-1) ELISA Kit
Product Code:RTFI00299
Size:96 Assays
Target:Rat P4ha1
Alias:P4ha1, PHalphaI
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 P4ha1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat P4ha1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)88-10294
EDTA plasma(n=5)86-10290
UFH plasma(n=5)87-10297
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat P4ha1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)89-102%85-98%85-105%
EDTA plasma(n=5)83-94%82-98%84-98%
UFH plasma(n=5)80-93%80-98%84-95%
Intra-Assay:CV <8%
Inter-Assay:CV <10%
Uniprot:P54001
UniProt Protein Function:Catalyzes the post-translational formation of 4-hydroxyproline in -Xaa-Pro-Gly- sequences in collagens and other proteins.
NCBI Summary:catalyzes the hydroxylation of -X-Pro-Gly- triplets to form hydroxyproline [RGD, Feb 2006]
UniProt Code:P54001
NCBI GenInfo Identifier:51036657
NCBI Gene ID:64475
NCBI Accession:NP_742059.2
UniProt Related Accession:P54001
Molecular Weight:
NCBI Full Name:prolyl 4-hydroxylase subunit alpha-1
NCBI Synonym Full Names:prolyl 4-hydroxylase subunit alpha 1
NCBI Official Symbol:P4ha1  
NCBI Official Synonym Symbols:PHalphaI  
NCBI Protein Information:prolyl 4-hydroxylase subunit alpha-1
UniProt Protein Name:Prolyl 4-hydroxylase subunit alpha-1
UniProt Synonym Protein Names:Procollagen-proline,2-oxoglutarate-4-dioxygenase subunit alpha-1
Protein Family:Antitoxin
UniProt Gene Name:P4ha1  
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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