Human UBR4 ELISA Kit (HUEB0470)- High Sensitivity

Product Name:	Human E3 ubiquitin-protein ligase UBR4 (UBR4) ELISA Kit
SKU:	HUEB0470
Size:	96T
Target:	Human E3 ubiquitin-protein ligase UBR4 (UBR4)
Synonyms:	600 kDa retinoblastoma protein-associated factor, N-recognin-4, RING-type E3 ubiquitin transferase UBR4, Retinoblastoma-associated factor of 600 kDa, Zinc finger UBR1-type protein 1, RBAF600, KIAA0462, KIAA1307, RBAF600, ZUBR1
Assay Type:	Competitive
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.625-40ng/mL
Sensitivity:	0.22ng/mL

Intra CV:

5.2%

Inter CV:

8.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	103-113%	109-120%	111-120%	90-90%
EDTA Plasma(N=5)	109-117%	105-115%	87-97%	107-119%
Heparin Plasma(N=5)	86-96%	95-104%	91-100%	110-120%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	97	91-103
Plasma	99	93-105

Function:

E3 ubiquitin-protein ligase which is a component of the N-end rule pathway. Recognizes and binds to proteins bearing specific N-terminal residues that are destabilizing according to the N-end rule, leading to their ubiquitination and subsequent degradation. Together with clathrin, forms meshwork structures involved in membrane morphogenesis and cytoskeletal organization. Regulates integrin-mediated signaling. May play a role in activation of FAK in response to cell-matrix interactions. Mediates ubiquitination of ACLY, leading to its subsequent degradation.

Uniprot:	Q5T4S7
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human E3 ubiquitin-protein ligase UBR4
Sub Unit:	Interacts with RB1 and calmodulin. Interacts with protein E7 from papilloma virus HPV-16, HPV-6B and HPV-11.
Subcellular Location:	Membrane Multi-pass membrane protein Cytoplasm Cytoplasm Cytoskeleton Nucleus Concentrates at the leading edge of membrane structures involved in actin motility.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	UBR4: E3 ubiquitin-protein ligase which is a component of the N-end rule pathway. Recognizes and binds to proteins bearing specific N-terminal residues that are destabilizing according to the N-end rule, leading to their ubiquitination and subsequent degradation. Together with clathrin, forms meshwork structures involved in membrane morphogenesis and cytoskeletal organization. Regulates integrin-mediated signaling. May play a role in activation of FAK in response to cell-matrix interactions. Belongs to the UBR4 family. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 6.3.2.-; EC 6.3.2.19; Ligase; Membrane protein, integral; Membrane protein, multi-pass; Ubiquitin conjugating system; Ubiquitin ligase Chromosomal Location of Human Ortholog: 1p36.13 Cellular Component: centrosome; cytosol; membrane; nucleoplasm; plasma membrane Molecular Function:protein binding; ubiquitin-protein ligase activity Biological Process: neutrophil degranulation; protein ubiquitination during ubiquitin-dependent protein catabolic process
NCBI Summary:	The protein encoded by this gene is an E3 ubiquitin-protein ligase that interacts with the retinoblastoma-associated protein in the nucleus and with calcium-bound calmodulin in the cytoplasm. The encoded protein appears to be a cytoskeletal component in the cytoplasm and part of the chromatin scaffold in the nucleus. In addition, this protein is a target of the human papillomavirus type 16 E7 oncoprotein. [provided by RefSeq, Aug 2010]
UniProt Code:	Q5T4S7
NCBI GenInfo Identifier:	74744979
NCBI Gene ID:	23352
NCBI Accession:	Q5T4S7.1
UniProt Secondary Accession:	Q5T4S7,O60646, O75050, Q4QRK5, Q5T4S8, Q5T4S9, Q5TBN8 Q5TBP2, Q6DKH8, A8MPT2, A8MQ33, A8MQB1,
UniProt Related Accession:	Q5T4S7
Molecular Weight:	574kDa
NCBI Full Name:	E3 ubiquitin-protein ligase UBR4
NCBI Synonym Full Names:	ubiquitin protein ligase E3 component n-recognin 4
NCBI Official Symbol:	UBR4
NCBI Official Synonym Symbols:	p600; ZUBR1; RBAF600
NCBI Protein Information:	E3 ubiquitin-protein ligase UBR4
UniProt Protein Name:	E3 ubiquitin-protein ligase UBR4
UniProt Synonym Protein Names:	600 kDa retinoblastoma protein-associated factor; N-recognin-4; RING-type E3 ubiquitin transferase UBR4; Retinoblastoma-associated factor of 600 kDa; RBAF600; p600; Zinc finger UBR1-type protein 1
Protein Family:	E3 ubiquitin-protein ligase
UniProt Gene Name:	UBR4

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 UBR4 / E3 ubiquitin-protein ligase ELISA Kit	Anti-UBR4 Antibody (CAB12193)
Human UBR4 (Ubiquitin Protein Ligase E3 Component N-Recognin 4) CLIA Kit (HUES01227)