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Sumo1 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00875
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

Sumo1 Colorimetric Cell-Based ELISA Kit

The SUMO1 Colorimetric Cell-Based ELISA Kit is a powerful tool for the precise measurement of SUMO1 levels in cell lysates and tissue homogenates. This kit offers exceptional sensitivity and accuracy, allowing for reliable and consistent results in various research applications.SUMO1, also known as Small Ubiquitin-like Modifier 1, is a key player in the regulation of protein function and cellular processes such as DNA repair, transcriptional regulation, and protein stability. Dysregulation of SUMO1 has been linked to numerous diseases, including cancer, neurodegenerative disorders, and immune dysfunction, making it a valuable target for investigating disease mechanisms and potential therapeutic interventions.

With its user-friendly protocols and high-quality components, the SUMO1 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers studying SUMOylation pathways and their impact on cellular function and disease progression. Trust in this kit to deliver accurate and insightful data for your research needs.

Product Name:Sumo1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00875
ELISA Type:Cell-Based
Target:Sumo1
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Sumo1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Sumo1 protein expression profile in cells. The kit can be used for measuring the relative amounts of Sumo1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Sumo1.

Qualitative determination of Sumo1 concentration is achieved by an indirect ELISA format. In essence, Sumo1 is captured by Sumo1-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 7341, UniProt ID: P63165, OMIM: 601912, Unigene: Hs.596171/Hs.81424
Gene Symbol:SUMO1
Sub Type:None
UniProt Protein Function:SUMO1: Ubiquitin-like protein that can be covalently attached to proteins as a monomer or a lysine-linked polymer. Covalent attachment via an isopeptide bond to its substrates requires prior activation by the E1 complex SAE1-SAE2 and linkage to the E2 enzyme UBE2I, and can be promoted by E3 ligases such as PIAS1-4, RANBP2 or CBX4. This post-translational modification on lysine residues of proteins plays a crucial role in a number of cellular processes such as nuclear transport, DNA replication and repair, mitosis and signal transduction. Involved for instance in targeting RANGAP1 to the nuclear pore complex protein RANBP2. Polymeric SUMO1 chains are also susceptible to polyubiquitination which functions as a signal for proteasomal degradation of modified proteins. May also regulate a network of genes involved in palate development. Interacts with SAE2, UBE2I, RANBP2, PIAS1 and PIAS2. Interacts with PARK2. Covalently attached to a number of proteins such as IKFZ1, PML, RANGAP1, HIPK2, SP100, p53, p73-alpha, MDM2, JUN, DNMT3B and TDG. Also interacts with HIF1A, HIPK2, HIPK3, CHD3, EXOSC9, RAD51 and RAD52. Interacts with USP25 (via ts SIM domain); the interaction weakly sumoylates USP25. Belongs to the ubiquitin family. SUMO subfamily.
UniProt Protein Details:

Protein type:Nuclear receptor co-regulator; Ubiquitin-like modifier

Chromosomal Location of Human Ortholog: 2q33

Cellular Component: nucleoplasm; PML body; nuclear membrane; dendrite; cytoplasm; nucleolus; fibrillar center; synapse; nuclear speck; nuclear pore; nucleus

Molecular Function:protein binding; ubiquitin protein ligase binding; transcription factor binding; SUMO ligase activity

Biological Process: negative regulation of transcription factor activity; cytokine and chemokine mediated signaling pathway; PML body organization and biogenesis; palate development; post-translational protein modification; DNA repair; negative regulation of DNA binding; regulation of protein localization; positive regulation of protein complex assembly; cellular protein metabolic process; protein sumoylation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; negative regulation of transcription, DNA-dependent

Disease: Orofacial Cleft 10

NCBI Summary:This gene encodes a protein that is a member of the SUMO (small ubiquitin-like modifier) protein family. It functions in a manner similar to ubiquitin in that it is bound to target proteins as part of a post-translational modification system. However, unlike ubiquitin which targets proteins for degradation, this protein is involved in a variety of cellular processes, such as nuclear transport, transcriptional regulation, apoptosis, and protein stability. It is not active until the last four amino acids of the carboxy-terminus have been cleaved off. Several pseudogenes have been reported for this gene. Alternate transcriptional splice variants encoding different isoforms have been characterized. [provided by RefSeq, Jul 2008]
UniProt Code:P63165
NCBI GenInfo Identifier:52783799
NCBI Gene ID:7341
NCBI Accession:P63165.1
UniProt Secondary Accession:P63165,P55856, Q6FGG0, Q6NZ62, Q93068, A8MUS8, B2R4I5
UniProt Related Accession:P63165
Molecular Weight:101
NCBI Full Name:Small ubiquitin-related modifier 1
NCBI Synonym Full Names:small ubiquitin-like modifier 1
NCBI Official Symbol:SUMO1  
NCBI Official Synonym Symbols:DAP1; GMP1; PIC1; SMT3; UBL1; OFC10; SENP2; SMT3C; SMT3H3  
NCBI Protein Information:small ubiquitin-related modifier 1; sentrin; SMT3 homolog 3; GAP modifying protein 1; ubiquitin-like protein UBL1; ubiquitin-like protein SMT3C; SMT3 suppressor of mif two 3 homolog 1; ubiquitin-homology domain protein PIC1
UniProt Protein Name:Small ubiquitin-related modifier 1
UniProt Synonym Protein Names:GAP-modifying protein 1; GMP1; SMT3 homolog 3; Sentrin; Ubiquitin-homology domain protein PIC1; Ubiquitin-like protein SMT3C; Smt3C; Ubiquitin-like protein UBL1
Protein Family:Small ubiquitin-related modifier
UniProt Gene Name:SUMO1  
UniProt Entry Name:SUMO1_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies (Anti-Sumo1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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