Human Ubiquilin-2 (UBQLN2) ELISA Kit- High Sensitivity

Product Name:	Human Ubiquilin-2 (UBQLN2) ELISA Kit
SKU:	HUEB0697
Size:	96T
Target:	Human Ubiquilin-2 (UBQLN2)
Synonyms:	Chap1, DSK2 homolog, Protein linking IAP with cytoskeleton 2, Ubiquitin-like product Chap1/Dsk2, PLIC-2, HRIHFB2157, N4BP4, PLIC2
Assay Type:	Competitive
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	78-5000pg/mL
Sensitivity:	34pg/mL

Intra CV:

5.3%

Inter CV:

10.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	94-103%	99-109%	108-118%	84-96%
EDTA Plasma(N=5)	97-108%	98-107%	100-110%	102-112%
Heparin Plasma(N=5)	100-109%	93-103%	92-104%	109-118%

Recovery:

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

Function:

Plays an important role in the regulation of different protein degradation mechanisms and pathways including ubiquitin-proteasome system (UPS), autophagy and the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Mediates the proteasomal targeting of misfolded or accumulated proteins for degradation by binding (via UBA domain) to their polyubiquitin chains and by interacting (via ubiquitin-like domain) with the subunits of the proteasome (PubMed:10983987). Plays a role in the ERAD pathway via its interaction with ER-localized proteins FAF2/UBXD8 and HERPUD1 and may form a link between the polyubiquitinated ERAD substrates and the proteasome (PubMed:24215460, PubMed:18307982). Involved in the regulation of macroautophagy and autophagosome formation; required for maturation of autophagy-related protein LC3 from the cytosolic form LC3-I to the membrane-bound form LC3-II and may assist in the maturation of autophagosomes to autolysosomes by mediating autophagosome-lysosome fusion (PubMed:19148225, PubMed:20529957). Negatively regulates the endocytosis of GPCR receptors: AVPR2 and ADRB2, by specifically reducing the rate at which receptor-arrestin complexes concentrate in clathrin-coated pits (CCPs) (PubMed:18199683).

Uniprot:	Q9UHD9
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Ubiquilin-2
Sub Unit:	Homodimer. Forms heterodimer with UBQLN1. Binds UBE3A and BTRC. Interacts with the 19S proteasome subunit. Interacts with C9orf72. Interacts with HNRNPA1 and HNRNPU. Found in a complex with UBQLN1 and MAP1LC3A/B/C. Interacts with EPS15, EPN1 and EPN2. Interacts with HERPUD1. Interacts with RAD23A. Interacts with TARDBP. Interacts (via C-terminus) with FAF2 (via N-terminus). Interacts with UBQLN4. Binds CD47.
Research Area:	Cell Biology
Subcellular Location:	Cytoplasm Nucleus Membrane Cytoplasmic vesicle Autophagosome Colocalizes with a subset of proteasomes, namely those that are cytoskeleton associated or free in the cytosol. Associated with fibers in mitotic cells.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	UBQLN2: Increases the half-life of proteins destined to be degraded by the proteasome; may modulate proteasome-mediated protein degradation. Defects in UBQLN2 are the cause of amyotrophic lateral sclerosis type 15 with or without frontotemporal dementia (ALS15). A neurodegenerative disorder affecting upper motor neurons in the brain and lower motor neurons in the brain stem and spinal cord, resulting in fatal paralysis. Sensory abnormalities are absent. The pathologic hallmarks of the disease include pallor of the corticospinal tract due to loss of motor neurons, presence of ubiquitin-positive inclusions within surviving motor neurons, and deposition of pathologic aggregates. The etiology of amyotrophic lateral sclerosis is likely to be multifactorial, involving both genetic and environmental factors. The disease is inherited in 5-10% of the cases. Patients with ALS15 may develop frontotemporal dementia.
UniProt Protein Details:	Protein type:Ubiquitin conjugating system Chromosomal Location of Human Ortholog: Xp11.21 Cellular Component: cytoplasm; plasma membrane Molecular Function:protein binding Biological Process: ER-associated protein catabolic process; regulation of macroautophagy Disease: Amyotrophic Lateral Sclerosis 15, With Or Without Frontotemporal Dementia
NCBI Summary:	This gene encodes an ubiquitin-like protein (ubiquilin) that shares high degree of similarity with related products in yeast, rat and frog. Ubiquilins contain a N-terminal ubiquitin-like domain and a C-terminal ubiquitin-associated domain. They physically associate with both proteasomes and ubiquitin ligases; and thus, are thought to functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation. This ubiquilin has also been shown to bind the ATPase domain of the Hsp70-like Stch protein. [provided by RefSeq, Oct 2009]
UniProt Code:	Q9UHD9
NCBI GenInfo Identifier:	124056593
NCBI Gene ID:	29978
NCBI Accession:	Q9UHD9.2
UniProt Secondary Accession:	Q9UHD9,O94798, Q5D027, Q9H3W6, Q9HAZ4,
UniProt Related Accession:	Q9UHD9
Molecular Weight:	65,696 Da
NCBI Full Name:	Ubiquilin-2
NCBI Synonym Full Names:	ubiquilin 2
NCBI Official Symbol:	UBQLN2
NCBI Official Synonym Symbols:	DSK2; ALS15; CHAP1; N4BP4; PLIC2; HRIHFB2157
NCBI Protein Information:	ubiquilin-2
UniProt Protein Name:	Ubiquilin-2
UniProt Synonym Protein Names:	Chap1; DSK2 homolog; Protein linking IAP with cytoskeleton 2; PLIC-2; hPLIC-2; Ubiquitin-like product Chap1/Dsk2
Protein Family:	Ubiquilin
UniProt Gene Name:	UBQLN2
UniProt Entry Name:	UBQL2_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

Human UBQLN2 / Ubiquilin-2 ELISA Kit