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Human PDCD4 ELISA Kit

SKU:
HUFI01876
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q53EL6
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich
Synonyms:
PDCD4, H731, nuclear antigen H731, Neoplastic transformation inhibitor protein, Nuclear antigen H731-like, programmed cell death 4, neoplastic transformation inhibitor, programmed cell death protein 4, Protein 197, 15a
Reactivity:
Human
Research Area:
Cell Death
€599
Frequently bought together:

Description

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Human PDCD4 ELISA

PDCD4 is a tumor suppressor and encodes a protein that binds to the eukaryotic translation initiation factor 4A1 and inhibits its function by preventing RNA binding. PDCD4 may also play a role in apoptosis. Diseases associated with PDCD4 include Colorectal Cancer and Tongue Squamous Cell Carcinoma. The Assay Genie Human PDCD4 ELISA is a highly sensitive assay for the quantitative measurement of PDCD4 in serum, blood, plasma, cell culture supernatant and tissue samples.

Product Name:Human PDCD4 ELISA Kit
Product Code:HUFI01876
Size:96 Assays
Alias:PDCD4, H731, nuclear antigen H731, Neoplastic transformation inhibitor protein, Nuclear antigen H731-like, programmed cell death 4, neoplastic transformation inhibitor, programmed cell death protein 4, Protein 197, 15a
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human PDCD4 concentrations in serum plasma and other biological fluids.
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 Human PDCD4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human PDCD4 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)92-10598
EDTA plasma(n=5)89-10497
UFH plasma(n=5)86-10092
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PDCD4 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%88-102%86-104%
EDTA plasma(n=5)85-96%86-97%82-100%
UFH plasma(n=5)80-96%80-90%80-90%
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
UniprotQ53EL6
UniProt Protein Function:PDCD4: protein localized to the nucleus in proliferating cells that seems to possess a tumor suppressor activity. Directly interacts with the RNA helicase eIF4A and inhibits protein synthesis by interfering with the assembly of the cap-dependent translation initiation complex. Suppresses carbonic anhydrase type II protein expression in carcinoid cell lines. Since tumor cells require a high bicarbonate flux for their growth, carbonic anhydrase suppression results in growth inhibition. Expression of this gene is modulated by cytokines in natural killer and T cells. The gene product is thought to play a role in apoptosis but the specific role has not yet been determined. Two differentially spliced isoforms have been identified.
UniProt Protein Details:Protein type:Tumor suppressor; Apoptosis

Chromosomal Location of Human Ortholog: 10q24

Cellular Component: cytoplasm; cytosol; nucleoplasm; nucleus

Molecular Function:protein binding; RNA binding

Biological Process: apoptosis; cell aging; negative regulation of apoptosis; negative regulation of cell cycle; negative regulation of JNK activity; negative regulation of transcription, DNA-dependent

NCBI Summary:This gene is a tumor suppressor and encodes a protein that binds to the eukaryotic translation initiation factor 4A1 and inhibits its function by preventing RNA binding. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Dec 2010]
UniProt Code:Q53EL6
NCBI GenInfo Identifier:317373317
NCBI Gene ID:27250
NCBI Accession:Q53EL6.2
UniProt Secondary Accession:Q53EL6,O15501, Q5VZS6, Q6PJI5, Q8TAR5, Q99834, B2RCV4 B5ME91,
UniProt Related Accession:Q53EL6
Molecular Weight:50,576 Da
NCBI Full Name:Programmed cell death protein 4
NCBI Synonym Full Names:programmed cell death 4 (neoplastic transformation inhibitor)
NCBI Official Symbol:PDCD4
NCBI Official Synonym Symbols:H731
NCBI Protein Information:programmed cell death protein 4
UniProt Protein Name:Programmed cell death protein 4
UniProt Synonym Protein Names:Neoplastic transformation inhibitor protein; Nuclear antigen H731-like; Protein 197/15a
Protein Family:Programmed cell death protein
UniProt Gene Name:PDCD4
UniProt Entry Name:PDCD4_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.

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