Human PDCD4 ELISA Kit (HUEB2349)- High Sensitivity

Product Name:	Human Programmed cell death protein 4 (PDCD4) ELISA Kit
SKU:	HUEB2349
Size:	96T
Target:	Human Programmed cell death protein 4 (PDCD4)
Synonyms:	Neoplastic transformation inhibitor protein, Nuclear antigen H731-like, Protein 197/15a, H731
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.13ng/mL

Intra CV:

4.0%

Inter CV:

7.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	102-113%	87-97%	102-111%	94-106%
EDTA Plasma(N=5)	108-118%	99-110%	112-112%	87-99%
Heparin Plasma(N=5)	108-118%	99-109%	103-113%	102-112%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	101	95-107
Plasma	103	97-109

Function:

Inhibits translation initiation and cap-dependent translation. May excert its function by hindering the interaction between EIF4A1 and EIF4G. Inhibits the helicase activity of EIF4A. Modulates the activation of JUN kinase. Down-regulates the expression of MAP4K1, thus inhibiting events important in driving invasion, namely, MAPK85 activation and consequent JUN-dependent transcription. May play a role in apoptosis. Tumor suppressor. Inhibits tumor promoter-induced neoplastic transformation. Binds RNA.

Uniprot:	Q53EL6
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Programmed cell death protein 4
Sub Unit:	Interacts (via MI domains) with EIF4A2 (By similarity). Interacts (via MI domains) with EIF4A1 (via N-terminal domain). Heterotrimer with EIF4A1; one molecule of PDCD4 binds two molecules of EIF4A1. Interacts with EIF4G1. May form a complex with EIF4A1 and EIF4G1. The interaction between PDCD4 and EIF4A1 interferes with the interaction between EIF4A1 and EIF4G. When phosphorylated, interacts with BTRC and FBXW11.
Research Area:	Cancer
Subcellular Location:	Nucleus Cytoplasm Shuttles between the nucleus and cytoplasm (By similarity). Predominantly nuclear under normal growth conditions, and when phosphorylated at Ser-457 (PubMed:16357133).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

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

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

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 -20°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 experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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 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.
Plasma	Collect 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 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 & 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 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.

ELISA	Antibodies
Human PDCD4 ELISA Kit	Anti-PDCD4 Antibody (CAB2420)
PDCD4 Colorimetric Cell-Based ELISA Kit	Anti-PDCD4 Antibody (CAB2570)[KO Validated]
PDCD4 (Phospho-Ser67) Colorimetric Cell-Based ELISA Kit	Anti-PDCD4 Antibody (CAB9068)
PDCD4 (Phospho-Ser457) Colorimetric Cell-Based ELISA Kit	PDCD4 Antibody (PACO01307)
	Phospho-PDCD4 (S457) Antibody (PACO02785)