Human PAP (Prostatic acid phosphatase) ELISA Kit (HUFI04691)

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SKU:
HUFI04691
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P15309
Sensitivity:
37.5pg/ml
Range:
62.5-4000pg/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
acid phosphatase, prostate, ACP 3, ACP3, ACPP, PAP, Prostatic acid phosphatase
Reactivity:
Human
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Description

Product Name:Human PAP (Prostatic acid phosphatase) ELISA Kit
Product Code:HUFI04691
Size:96 Assays
Alias:acid phosphatase ELISA Kit, prostate ELISA Kit, ACP 3 ELISA Kit, ACP3 ELISA Kit, ACPP ELISA Kit, PAP ELISA Kit, Prostatic acid phosphatase ELISA Kit
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human PAP (Prostatic acid phosphatase) concentrations in serum plasma and other biological fluids.
Sensitivity:< 37.5pg/ml
Range:62.5-4000pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human PAP (Prostatic acid phosphatase) and the recovery rates were calculated by comparing the measured value to the expected amount of Human PAP (Prostatic acid phosphatase) in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)89-10396
EDTA plasma(n=5)86-9993
heparin plasma(n=5)88-9893
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PAP (Prostatic acid phosphatase) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-102%85-102%85-102%
EDTA plasma(n=5)88-101%82-99%85-96%
UFH plasma(n=5)86-92%81-99%83-95%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniProt Protein Function:ACPP: A non-specific tyrosine phosphatase that dephosphorylates a diverse number of substrates under acidic conditions (pH 4-6) including alkyl, aryl, and acyl orthophosphate monoesters and phosphorylated proteins. Has lipid phosphatase activity and inactivates lysophosphatidic acid in seminal plasma. Belongs to the histidine acid phosphatase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:EC 3.1.3.2; Cofactor and Vitamin Metabolism - riboflavin; Phosphatase; EC 3.1.3.5; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 3q22.1

Cellular Component: multivesicular body; extracellular space; Golgi cisterna; apical part of cell; lysosomal membrane; plasma membrane; integral to membrane; vesicle membrane; intracellular; nucleus; secretory granule; filopodium

Molecular Function:5'-nucleotidase activity; choline binding; identical protein binding; acid phosphatase activity; protein homodimerization activity; phosphoric monoester hydrolase activity; thiamin phosphate phosphatase activity

Biological Process: thiamin metabolic process; dephosphorylation; positive regulation of adenosine receptor signaling pathway; nucleotide metabolic process; regulation of sensory perception of pain; adenosine metabolic process; purine base metabolic process

NCBI Summary:This gene encodes an enzyme that catalyzes the conversion of orthophosphoric monoester to alcohol and orthophosphate. It is synthesized under androgen regulation and is secreted by the epithelial cells of the prostate gland. An alternatively spliced transcript variant encoding a longer isoform has been found for this gene. This isoform contains a transmembrane domain and is localized in the plasma membrane-endosomal-lysosomal pathway. [provided by RefSeq, Sep 2008]
UniProt Code:P15309
NCBI GenInfo Identifier:130730
NCBI Gene ID:55
NCBI Accession:P15309.3
UniProt Related Accession:P15309
Molecular Weight:
NCBI Full Name:Prostatic acid phosphatase
NCBI Synonym Full Names:acid phosphatase, prostate
NCBI Official Symbol:ACPP
NCBI Official Synonym Symbols:ACP3; 5'-NT; ACP-3
NCBI Protein Information:prostatic acid phosphatase
UniProt Protein Name:Prostatic acid phosphatase
UniProt Synonym Protein Names:5'-nucleotidase (EC:3.1.3.5); 5'-NT; Ecto-5'-nucleotidase; Thiamine monophosphatase; TMPase
UniProt Gene Name:ACPP
UniProt Entry Name:PPAP_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
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 TypeProtocol
SerumIf 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 clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 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 lysatesRinse 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 MilkCollect 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.
AntibodiesELISA Kits
Anti-ACPP Antibody (CAB13457)Human ACP3/PAP (Prostatic Acid Phosphatase 3) CLIA Kit (HUES01021)
Anti-ACPP Antibody (CAB1937)
ACPP Antibody (PACO07569)
ACPP Antibody (PACO13816)
ACPP Antibody (PACO13817)
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