The Mouse NAD-dependent deacetylase sirtuin-6 (SIRT6) ELISA Kit is a highly sensitive and specific assay designed for the accurate measurement of SIRT6 levels in mouse serum, plasma, and cell culture supernatants. With its superior precision and reliability, this kit is an invaluable tool for researchers conducting studies related to aging, metabolic disorders, and cellular stress response pathways.SIRT6 is a key regulator of various cellular processes, including DNA repair, glucose metabolism, and inflammation. Dysregulation of SIRT6 has been implicated in several age-related diseases, such as diabetes, cardiovascular diseases, and neurodegenerative disorders.
By accurately quantifying SIRT6 levels, researchers can gain insight into its role in disease pathogenesis and potentially identify new therapeutic targets for intervention.Trust in the Mouse NAD-dependent deacetylase sirtuin-6 (SIRT6) ELISA Kit for precise and reproducible results that will advance your research and contribute to the understanding of SIRT6 biology in health and disease.
NAD-dependent protein deacetylase. Has deacetylase activity towards 'Lys-9' and 'Lys-56' of histone H3. Modulates acetylation of histone H3 in telomeric chromatin during the S-phase of the cell cycle. Deacetylates 'Lys-9' of histone H3 at NF-kappa-B target promoters and may down-regulate the expression of a subset of NF-kappa-B target genes. Deacetylation of nucleosomes interferes with RELA binding to target DNA. May be required for the association of WRN with telomeres during S-phase and for normal telomere maintenance. Required for genomic stability. Required for normal IGF1 serum levels and normal glucose homeostasis. Modulates cellular senescence and apoptosis. Regulates the production of TNF protein.
Uniprot:
P59941
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse NAD-dependent deacetylase sirtuin-6
Research Area:
Epigenetics
Subcellular Location:
Nucleus Predominantly nuclear. Associated with chromatin.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
SIRT6: NAD-dependent protein deacetylase. Has deacetylase activity towards histone H3K9Ac and H3K56Ac. Modulates acetylation of histone H3 in telomeric chromatin during the S-phase of the cell cycle. Deacetylates histone H3K9Ac at NF-kappa-B target promoters and may down-regulate the expression of a subset of NF- kappa-B target genes. Acts as a corepressor of the transcription factor HIF1A to control the expression of multiple glycolytic genes to regulate glucose homeostasis. Required for genomic stability. Regulates the production of TNF protein. Has a role in the regulation of life span. Deacetylation of nucleosomes interferes with RELA binding to target DNA. May be required for the association of WRN with telomeres during S-phase and for normal telomere maintenance. Required for genomic stability. Required for normal IGF1 serum levels and normal glucose homeostasis. Modulates cellular senescence and apoptosis. On DNA damage, promotes DNA end resection via deacetylation of RBBP8. Has very weak deacetylase activity and can bind NAD(+) in the absence of acetylated substrate. Interacts with RELA. Interacts with RBBP8; the interaction deacetylates RBBP8. Belongs to the sirtuin family. Class IV subfamily. 3 isoforms of the human protein are produced by alternative splicing.Protein type: Deacetylase; EC 2.4.2.31; EC 3.5.1.-; TransferaseChromosomal Location of Human Ortholog: 10 C1|10 39.72 cMCellular Component: cytoplasm; nuclear telomeric heterochromatin; nucleoplasm; nucleusMolecular Function: chromatin binding; hydrolase activity; metal ion binding; NAD(P)+-protein-arginine ADP-ribosyltransferase activity; NAD-dependent histone deacetylase activity (H3-K9 specific); protein deacetylase activity; transcription corepressor activityBiological Process: base-excision repair; glucose homeostasis; negative regulation of cell proliferation; negative regulation of glucose import; negative regulation of glycolytic process; negative regulation of transcription, DNA-dependent; positive regulation of chromatin silencing at telomere; positive regulation of fibroblast proliferation; positive regulation of telomere maintenance; protein ADP-ribosylation; protein destabilization
NAD-dependent protein deacetylase sirtuin-6 isoform 1
NCBI Synonym Full Names:
sirtuin 6
NCBI Official Symbol:
Sirt6
NCBI Official Synonym Symbols:
Sir2l6; AI043036; 2810449N18Rik
NCBI Protein Information:
NAD-dependent protein deacetylase sirtuin-6
UniProt Protein Name:
NAD-dependent protein deacetylase sirtuin-6
UniProt Synonym Protein Names:
Regulatory protein SIR2 homolog 6; SIR2-like protein 6
Protein Family:
NAD-dependent protein deacetylase
UniProt Gene Name:
Sirt6
UniProt Entry Name:
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.
