Human DDOST ELISA Kit (HUEB2166)- High Sensitivity

Product Name:	Human Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit (DDOST) ELISA Kit
Product Code:	HUEB2166
Alias:	Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit, DDOST 48 kDa subunit, Oligosaccharyl transferase 48 kDa subunit, DDOST, KIAA0115, OST48, OK/SW-cl.45, 2.4.99.18
Uniprot:	P39656
Reactivity:	Human
Range:	0.312-20 ng/mL
Detection Method:	Sandwich
Size:	96 Assay
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	DDOST: Essential subunit of N-oligosaccharyl transferase enzyme which catalyzes the transfer of a high mannose oligosaccharide to an asparagine residue within an Asn-X-Ser/Thr consensus motif in nascent polypeptide chains. Defects in DDOST are the cause of congenital disorder of glycosylation type 1R (CDG1R). A multisystem disorder caused by a defect in glycoprotein biosynthesis and characterized by under-glycosylated serum glycoproteins. Congenital disorders of glycosylation result in a wide variety of clinical features, such as defects in the nervous system development, psychomotor retardation, dysmorphic features, hypotonia, coagulation disorders, and immunodeficiency. The broad spectrum of features reflects the critical role of N-glycoproteins during embryonic development, differentiation, and maintenance of cell functions. Belongs to the DDOST 48 kDa subunit family.
UniProt Protein Details:	Protein type:EC 2.4.99.18; Glycan Metabolism - N-glycan biosynthesis; Membrane protein, integral; Endoplasmic reticulum; Transferase Chromosomal Location of Human Ortholog: 1p36.1 Cellular Component: endoplasmic reticulum membrane; protein complex; intracellular membrane-bound organelle; membrane; endoplasmic reticulum; oligosaccharyl transferase complex; integral to membrane Molecular Function:protein binding; dolichyl-diphosphooligosaccharide-protein glycotransferase activity; oligosaccharyl transferase activity Biological Process: SRP-dependent cotranslational protein targeting to membrane; cellular protein metabolic process; T cell activation; translation; response to cytokine stimulus; protein amino acid N-linked glycosylation; protein amino acid glycosylation; innate immune response; gene expression; protein amino acid N-linked glycosylation via asparagine; post-translational protein modification Disease: Congenital Disorder Of Glycosylation, Type Ir
NCBI Summary:	This gene encodes a component of the oligosaccharyltransferase complex which catalyzes the transfer of high-mannose oligosaccharides to asparagine residues on nascent polypeptides in the lumen of the rough endoplasmic reticulum. The protein complex co-purifies with ribosomes. The product of this gene is also implicated in the processing of advanced glycation endproducts (AGEs), which form from non-enzymatic reactions between sugars and proteins or lipids and are associated with aging and hyperglycemia. [provided by RefSeq, Jul 2008]
UniProt Code:	P39656
NCBI GenInfo Identifier:	239938926
NCBI Gene ID:	1650
NCBI Accession:	P39656.4
UniProt Secondary Accession:	P39656,O43244, Q5VWA5, Q8NI93, Q9BUI2, B2RDQ4, B4DJE3 B4DLI2,
UniProt Related Accession:	P39656
Molecular Weight:	49,020 Da
NCBI Full Name:	Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit
NCBI Synonym Full Names:	dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit (non-catalytic)
NCBI Official Symbol:	DDOST
NCBI Official Synonym Symbols:	OST; WBP1; AGER1; CDG1R; OST48; OKSWcl45
NCBI Protein Information:	dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit; oligosaccharyltransferase subunit 48; advanced glycation endproduct receptor 1; oligosaccharyltransferase 48 kDa subunit; oligosaccharyl transferase 48 kDa subunit; dolichyl-diphosphooligosaccharide-protein glycotransferase
UniProt Protein Name:	Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit
Protein Family:	Dolichyl-diphosphooligosaccharide--protein glycosyltransferase
UniProt Gene Name:	DDOST
UniProt Entry Name:	OST48_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 DDOST ELISA Kit