Rat Protein argonaute-2 (Eif2c2) ELISA Kit- High Sensitivity

Product Name:	Rat Protein argonaute-2 (Eif2c2) ELISA Kit
SKU:	RTEB0699
Size:	96T
Target:	Rat Protein argonaute-2 (Eif2c2)
Synonyms:	Argonaute RISC catalytic component 2, Eukaryotic translation initiation factor 2C 2, Golgi ER protein 95 kDa, Protein slicer, eIF-2C 2, GERp95, 3.1.26.n2, Argonaute2, Eif2c2
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	15.6-1000pg/mL
Sensitivity:	7.4pg/mL

Intra CV:	Provided with the Kit
Inter CV:	Provided with the Kit
Linearity:	Provided with the Kit
Recovery:	Provided with the Kit
Function:	Required for RNA-mediated gene silencing (RNAi) by the RNA-induced silencing complex (RISC). The 'minimal RISC' appears to include AGO2 bound to a short guide RNA such as a microRNA (miRNA) or short interfering RNA (siRNA). These guide RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. The precise mechanism of gene silencing depends on the degree of complementarity between the miRNA or siRNA and its target. Binding of RISC to a perfectly complementary mRNA generally results in silencing due to endonucleolytic cleavage of the mRNA specifically by AGO2. Binding of RISC to a partially complementary mRNA results in silencing through inhibition of translation, and this is independent of endonuclease activity. May inhibit translation initiation by binding to the 7-methylguanosine cap, thereby preventing the recruitment of the translation initiation factor eIF4-E. May also inhibit translation initiation via interaction with EIF6, which itself binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The inhibition of translational initiation leads to the accumulation of the affected mRNA in cytoplasmic processing bodies (P-bodies), where mRNA degradation may subsequently occur. In some cases RISC-mediated translational repression is also observed for miRNAs that perfectly match the 3' untranslated region (3'-UTR). Can also up-regulate the translation of specific mRNAs under certain growth conditions. Binds to the AU element of the 3'-UTR of the TNF (TNF-alpha) mRNA and up-regulates translation under conditions of serum starvation. Also required for transcriptional gene silencing (TGS), in which short RNAs known as antigene RNAs or agRNAs direct the transcriptional repression of complementary promoter regions.

Uniprot:	Q9QZ81
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Protein argonaute-2
Sub Unit:	Interacts with DICER1 through its Piwi domain and with TARBP2 during assembly of the RNA-induced silencing complex (RISC). Together, DICER1, AGO2 and TARBP2 constitute the trimeric RISC loading complex (RLC), or micro-RNA (miRNA) loading complex (miRLC). Within the RLC/miRLC, DICER1 and TARBP2 are required to process precursor miRNAs (pre-miRNAs) to mature miRNAs and then load them onto AGO2. AGO2 bound to the mature miRNA constitutes the minimal RISC and may subsequently dissociate from DICER1 and TARBP2. Note however that the term RISC has also been used to describe the trimeric RLC/miRLC. The formation of RISC complexes containing siRNAs rather than miRNAs appears to occur independently of DICER1. Interacts with AGO1. Also interacts with DDB1, DDX5, DDX6, DDX20, DHX30, DHX36, DDX47, DHX9, ELAVL, FXR1, GEMIN4, HNRNPF, IGF2BP1, ILF3, IMP8, MATR3, PABPC1, PRMT5, P4HA1, P4HB, RBM4, SART3, TNRC6A, TNRC6B, UPF1 and YBX1. Interacts with the P-body components DCP1A and XRN1. Associates with polysomes and messenger ribonucleoproteins (mNRPs). Interacts with RBM4; the interaction is modulated under stress-induced conditions, occurs under both cell proliferation and differentiation conditions and in an RNA- and phosphorylation-independent manner. Interacts with LIMD1, WTIP and AJUBA. Interacts with TRIM71; the interaction increases in presence of RNA. Interacts with APOBEC3G in an RNA-dependent manner. Interacts with APOBEC3A, APOBEC3C, APOBEC3F and APOBEC3H. Interacts with DICER1, TARBP2, EIF6, MOV10 and RPL7A (60S ribosome subunit); they form a large RNA-induced silencing complex (RISC). Interacts with FMR1. Interacts with ZFP36. Interacts with RC3H1; the interaction is RNA independent (By similarity). Found in a complex, composed of AGO2, CHD7 and FAM172A (By similarity). Interacts with SND1 (By similarity).
Subcellular Location:	Cytoplasm P-body Nucleus Translational repression of mRNAs results in their recruitment to P-bodies. Translocation to the nucleus requires IMP8.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	AGO2: the endonuclease in RNA-induced silencing complexes (RISC) that cleaves siRNA/mRNA heteroduplexes bound to RISC. Ago2, along with Dicer and TRBP, are major components of RISC. Interacts with Dicer1 through its Piwi domain. Required for proper fibroblast growth factor signaling during gastrulation. Essential for embryonic development as well as RNA-mediated gene silencing (RNAi).Protein type: RNA processing; RNA-binding; EC 3.1.26.n2Chromosomal Location of Human Ortholog: 8q24Cellular Component: nucleoplasm; polysome; mRNA cap complex; membrane; cytoplasm; ribonucleoprotein complex; cytosolMolecular Function: mRNA binding; endoribonuclease activity; protein binding; miRNA binding; siRNA binding; translation initiation factor activity; metal ion binding; RNA 7-methylguanosine cap bindingBiological Process: epidermal growth factor receptor signaling pathway; negative regulation of translational initiation; Notch signaling pathway; fibroblast growth factor receptor signaling pathway; phosphoinositide-mediated signaling; nerve growth factor receptor signaling pathway; transcription, DNA-dependent; RNA interference, siRNA loading onto RISC; translation; miRNA-mediated gene silencing, mRNA cleavage; miRNA-mediated gene silencing, miRNA loading onto RISC; miRNA-mediated gene silencing, negative regulation of translation; post-embryonic development; regulation of transcription, DNA-dependent; pre-microRNA processing; translational initiation; innate immune response; RNA-mediated gene silencing; gene expression; posttranscriptional gene silencing
UniProt Protein Details:
NCBI Summary:	This gene encodes a member of the Argonaute family of proteins which play a role in RNA interference. The encoded protein is highly basic, and contains a PAZ domain and a PIWI domain. It may interact with dicer1 and play a role in short-interfering-RNA-mediated gene silencing. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2009]
UniProt Code:	Q9QZ81
NCBI GenInfo Identifier:	229463006
NCBI Gene ID:	27161
NCBI Accession:	Q9UKV8.3
UniProt Secondary Accession:	Q9QZ81,Q8CJG0, Q9QZ81
UniProt Related Accession:	Q9QZ81,Q9UKV8
Molecular Weight:	97kDa
NCBI Full Name:	Protein argonaute-2
NCBI Synonym Full Names:	argonaute RISC catalytic component 2
NCBI Official Symbol:	AGO2
NCBI Official Synonym Symbols:	PPD; Q10; CASC7; EIF2C2; LINC00980
NCBI Protein Information:	protein argonaute-2
UniProt Protein Name:	Protein argonaute-2
UniProt Synonym Protein Names:	Argonaute RISC catalytic component 2Eukaryotic translation initiation factor 2C 2; PAZ Piwi domain protein
Protein Family:	Protein argonaute
UniProt Gene Name:	AGO2
UniProt Entry Name:	AGO2_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.