Human Angiogenin (ANG) ELISA Kit- High Sensitivity

Product Name:	Human Angiogenin (ANG) ELISA Kit
SKU:	HUEB0100
Size:	96T
Target:	Human Angiogenin (ANG)
Synonyms:	Ribonuclease 5, RNase 5, RNASE5
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	15.6-1000pg/mL
Sensitivity:	5.7pg/mL

Intra CV:

5.9%

Inter CV:

9.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	81-92%	98-110%	103-112%	81-91%
EDTA Plasma(N=5)	107-117%	99-109%	100-109%	91-101%
Heparin Plasma(N=5)	90-98%	98-108%	107-119%	102-112%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	91	85-97
Plasma	93	87-99

Function:

Binds to actin on the surface of endothelial cells; once bound, angiogenin is endocytosed and translocated to the nucleus. Stimulates ribosomal RNA synthesis including that containing the initiation site sequences of 45S rRNA. Cleaves tRNA within anticodon loops to produce tRNA-derived stress-induced fragments (tiRNAs) which inhibit protein synthesis and triggers the assembly of stress granules (SGs). Angiogenin induces vascularization of normal and malignant tissues. Angiogenic activity is regulated by interaction with RNH1 in vivo.

Uniprot:	P03950
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Angiogenin
Sub Unit:	Homodimer (PubMed:25372031). Interacts with and forms a tight 1:1 complex with RNH1. Dimerization of two such complexes may occur.
Research Area:	Cardiovascular
Subcellular Location:	Cytoplasmic vesicle Secretory vesicle lumen Secreted Nucleus Nucleus Nucleolus Rapidly endocytosed by target cells and translocated to the nucleus where it accumulates in the nucleolus and binds to DNA (PubMed:12051708).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	ANG: May function as a tRNA-specific ribonuclease that abolishes protein synthesis by specifically hydrolyzing cellular tRNAs. Binds to actin on the surface of endothelial cells; once bound, angiogenin is endocytosed and translocated to the nucleus. Angiogenin induces vascularization of normal and malignant tissues. Angiogenic activity is regulated by interaction with RNH1 in vivo. Defects in ANG are the cause of susceptibility to amyotrophic lateral sclerosis type 9 (ALS9). ALS is a degenerative disorder of motor neurons in the cortex, brain stem and spinal cord. ALS is characterized by muscular weakness and atrophy. Belongs to the pancreatic ribonuclease family.
UniProt Protein Details:	Protein type:RNA-binding; Secreted, signal peptide; Ribonuclease; EC 3.1.27.-; Secreted; Motility/polarity/chemotaxis; Nucleolus; Extracellular matrix; Cell cycle regulation Chromosomal Location of Human Ortholog: 14q11.1-q11.2 Cellular Component: angiogenin-PRI complex; basal lamina; cell soma; cytoplasmic membrane-bound vesicle; extracellular region; extracellular space; growth cone; nucleolus; nucleus Molecular Function:actin binding; copper ion binding; DNA binding; endonuclease activity; heparin binding; peptide binding; protein binding; protein homodimerization activity; receptor binding; ribonuclease activity; rRNA binding Biological Process: actin filament polymerization; activation of phospholipase A2; activation of protein kinase B; angiogenesis; antibacterial humoral response; antifungal humoral response; cell communication; cell migration; defense response to Gram-positive bacterium; diacylglycerol biosynthetic process; homeostatic process; innate immune response; intercellular junction assembly and maintenance; negative regulation of smooth muscle cell proliferation; negative regulation of translation; oocyte maturation; ovarian follicle development; phospholipase C activation; placenta development; positive regulation of endothelial cell proliferation; positive regulation of phosphorylation; positive regulation of protein secretion; response to hormone stimulus; response to hypoxia; response to yeast; rRNA transcription Disease: Amyotrophic Lateral Sclerosis 9
NCBI Summary:	The protein encoded by this gene is an exceedingly potent mediator of new blood vessel formation. It hydrolyzes cellular tRNAs resulting in decreased protein synthesis and is similar to pancreatic ribonuclease. In addition, the mature peptide has antimicrobial activity against some bacteria and fungi, including S. pneumoniae and C. albicans. Alternative splicing results in two transcript variants encoding the same protein. This gene and the gene that encodes ribonuclease, RNase A family, 4 share promoters and 5' exons. Each gene splices to a unique downstream exon that contains its complete coding region. [provided by RefSeq, Aug 2014]
UniProt Code:	P03950
NCBI GenInfo Identifier:	113873
NCBI Gene ID:	283
NCBI Accession:	P03950.1
UniProt Secondary Accession:	P03950,Q05CV1, Q53X86, Q6P5T2, Q8WXE7,
UniProt Related Accession:	P03950
Molecular Weight:	16,550 Da
NCBI Full Name:	Angiogenin
NCBI Synonym Full Names:	angiogenin, ribonuclease, RNase A family, 5
NCBI Official Symbol:	ANG
NCBI Official Synonym Symbols:	ALS9; RAA1; HEL168; RNASE4; RNASE5
NCBI Protein Information:	angiogenin
UniProt Protein Name:	Angiogenin
UniProt Synonym Protein Names:	Ribonuclease 5; RNase 5
Protein Family:	Angiogenin
UniProt Gene Name:	ANG
UniProt Entry Name:	ANGI_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 Angiogenin / ANG ELISA Kit	Anti-ANG Antibody (CAB13465)
Human ANG (Angiogenin) CLIA Kit (HUES00006)	Anti-ANG Antibody (CAB1546)
Human ANG (Angiostatin) CLIA Kit (HUES00234)	ANG Antibody (PACO02209)
Human ANG (Angiostatin) ELISA Kit (HUES01546)	ANAPC5 Antibody (PACO07729)
	ALPL Antibody (PACO13839)