Human Immunology ELISA Kits 10

Human Monocarboxylate transporter 1 (SLC16A1) ELISA Kit

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SKU:
HUEB2534
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P53985
ELISA Type:
Sandwich
Reactivity:
Human

Description

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Product Name:Human Monocarboxylate transporter 1 (SLC16A1) ELISA Kit
Product Code:HUEB2534
Alias:Monocarboxylate transporter 1, MCT 1, Solute carrier family 16 member 1, SLC16A1, MCT1
Uniprot:P53985
Reactivity:Human
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:MCT1: Proton-linked monocarboxylate transporter. Catalyzes the rapid transport across the plasma membrane of many monocarboxylates such as lactate, pyruvate, branched-chain oxo acids derived from leucine, valine and isoleucine, and the ketone bodies acetoacetate, beta-hydroxybutyrate and acetate. Defects in SLC16A1 are the cause of symptomatic deficiency in lactate transport (SDLT); also known as erythrocyte lactate transporter defect. Deficiency of lactate transporter may result in an acidic intracellular environment created by muscle activity with consequent degeneration of muscle and release of myoglobin and creatine kinase. This defect might compromise extreme performance in otherwise healthy individuals. Defects in SLC16A1 are the cause of familial hyperinsulinemic hypoglycemia type 7 (HHF7); also known as exercise-induced hyperinsulinemic hypoglycemia. HHF7 is a dominantly inherited hypoglycemic disorder characterized by inappropriate insulin secretion during anaerobic exercise or on pyruvate load. Belongs to the major facilitator superfamily. Monocarboxylate porter (TC 2.A.1.13) family.
UniProt Protein Details:

Protein type:Transporter, SLC family; Membrane protein, multi-pass; Membrane protein, integral; Transporter; Mitochondrial

Chromosomal Location of Human Ortholog: 1p12

Cellular Component: centrosome; membrane; mitochondrion; integral to plasma membrane; integral to membrane; plasma membrane

Molecular Function:mevalonate transmembrane transporter activity; protein homodimerization activity; monocarboxylic acid transmembrane transporter activity; symporter activity; lactate transmembrane transporter activity; secondary active monocarboxylate transmembrane transporter activity

Biological Process: response to food; monocarboxylic acid transport; glucose homeostasis; cellular metabolic process; centrosome organization and biogenesis; mevalonate transport; lipid metabolic process; pyruvate metabolic process; blood coagulation; leukocyte migration; transmembrane transport; regulation of insulin secretion; behavioral response to nutrient

Disease: Erythrocyte Lactate Transporter Defect; Monocarboxylate Transporter 1 Deficiency; Hyperinsulinemic Hypoglycemia, Familial, 7

NCBI Summary:The protein encoded by this gene is a proton-linked monocarboxylate transporter that catalyzes the movement of many monocarboxylates, such as lactate and pyruvate, across the plasma membrane. Mutations in this gene are associated with erythrocyte lactate transporter defect. Alternatively spliced transcript variants have been found for this gene.[provided by RefSeq, Oct 2009]
UniProt Code:P53985
NCBI GenInfo Identifier:313104214
NCBI Gene ID:6566
NCBI Accession:P53985.3
UniProt Secondary Accession:P53985,Q49A45, Q5T8R6, Q9NSJ9,
UniProt Related Accession:P53985
Molecular Weight:46,234 Da
NCBI Full Name:Monocarboxylate transporter 1
NCBI Synonym Full Names:solute carrier family 16 (monocarboxylate transporter), member 1
NCBI Official Symbol:SLC16A1  
NCBI Official Synonym Symbols:MCT; HHF7; MCT1  
NCBI Protein Information:monocarboxylate transporter 1; MCT 1; solute carrier family 16 member 1; solute carrier family 16 (monocarboxylic acid transporters), member 1; solute carrier family 16, member 1 (monocarboxylic acid transporter 1)
UniProt Protein Name:Monocarboxylate transporter 1
UniProt Synonym Protein Names:Solute carrier family 16 member 1
Protein Family:Monocarboxylate transporter
UniProt Gene Name:SLC16A1  
UniProt Entry Name:MOT1_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.
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