Product Name:	Rat Alpha-1A adrenergic receptor (Adra1a) ELISA Kit
SKU:	RTEB0858
Size:	96T
Target:	Rat Alpha-1A adrenergic receptor (Adra1a)
Synonyms:	Alpha-1A adrenoreceptor, Alpha-1C adrenergic receptor, Alpha-1A adrenoceptor, Adra1c
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.156-10ng/mL
Sensitivity:	0.087ng/mL

Intra CV:

6.4%

Inter CV:

8.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	101-111%	106-116%	82-91%	81-91%
EDTA Plasma(N=5)	104-116%	87-96%	96-105%	101-111%
Heparin Plasma(N=5)	92-103%	104-113%	90-100%	106-118%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	103	97-109
Plasma	105	99-111

Function:

This alpha-adrenergic receptor mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system. Its effect is mediated by G(q) and G(11) proteins. Nuclear ADRA1A-ADRA1B heterooligomers regulate phenylephrine (PE)-stimulated ERK signaling in cardiac myocytes.

Uniprot:	P43140
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Alpha-1A adrenergic receptor
Sub Unit:	Homo- and heterooligomer. Heterooligomerizes with ADRA1B homooligomers in cardiac myocytes.
Research Area:	Cancer
Subcellular Location:	Nucleus membrane Multi-pass membrane protein Cell membrane Multi-pass membrane protein Location at the nuclear membrane facilitates heterooligomerization and regulates ERK-mediated signaling in cardiac myocytes. Colocalizes with GNAQ, PLCB1 as well as LAP2 at the nuclear membrane of cardiac myocytes (By similarity).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	ADRA1A: This alpha-adrenergic receptor mediates its action by association with G proteins that activate a phosphatidylinositol- calcium second messenger system. Its effect is mediated by G(q) and G(11) proteins. Nuclear ADRA1A-ADRA1B heterooligomers regulate phenylephrine(PE)-stimulated ERK signaling in cardiac myocytes. Belongs to the G-protein coupled receptor 1 family. Adrenergic receptor subfamily. ADRA1A sub-subfamily. 9 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, multi-pass; Membrane protein, integral; Receptor, GPCR; GPCR, family 1 Cellular Component: nuclear membrane; membrane; T-tubule; plasma membrane; integral to membrane; Z disc; nucleus Molecular Function:protein heterodimerization activity; alpha1-adrenergic receptor activity Biological Process: response to drug; adult heart development; positive regulation of the force of heart contraction by epinephrine-norepinephrine; positive regulation of synaptic transmission, GABAergic; response to hormone stimulus; positive regulation of heart rate; positive regulation of systemic arterial blood pressure; norepinephrine-epinephrine vasoconstriction involved in regulation of systemic arterial blood pressure; micturition; negative regulation of heart rate in baroreceptor response to increased systemic arterial blood pressure; organ growth; elevation of cytosolic calcium ion concentration; negative regulation of Rho protein signal transduction; positive regulation of MAPKKK cascade; phospholipase C activation; positive regulation of vasoconstriction; G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); positive regulation of action potential; positive regulation of smooth muscle contraction; cell growth; positive regulation of heart rate by epinephrine-norepinephrine; aging
NCBI Summary:	binds adrenergic compounds and analogs; activates phospholipase C mediated signal transduction [RGD, Feb 2006]
UniProt Code:	P43140
NCBI GenInfo Identifier:	148747171
NCBI Gene ID:	29412
NCBI Accession:	NP_058887.2
UniProt Related Accession:	P43140
Molecular Weight:	51,598 Da
NCBI Full Name:	alpha-1A adrenergic receptor
NCBI Synonym Full Names:	adrenoceptor alpha 1A
NCBI Official Symbol:	Adra1a
NCBI Official Synonym Symbols:	Adra1c
NCBI Protein Information:	alpha-1A adrenergic receptor
UniProt Protein Name:	Alpha-1A adrenergic receptor
UniProt Synonym Protein Names:	Alpha-1A adrenoreceptor; Alpha-1A adrenoceptor; Alpha-1C adrenergic receptor
Protein Family:	Alpha-1A adrenergic receptor
UniProt Gene Name:	Adra1a
UniProt Entry Name:	ADA1A_RAT

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.