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SMG7 Colorimetric Cell-Based ELISA

SKU:
CBCAB01168
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Detection Method:
Colorimetric
$719
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Description

system_update_altDatasheetMSDS

SMG7 Colorimetric Cell-Based ELISA

The SMG7 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for the detection and quantification of SMG7 protein levels in cell lysates. This kit offers high sensitivity and accuracy, allowing for precise measurements of SMG7 levels in various cell types and experimental conditions.SMG7 is a critical protein involved in the regulation of mRNA degradation and quality control processes in the cell. Dysregulation of SMG7 has been linked to various diseases, including cancer, neurodegenerative disorders, and autoimmune conditions.

By accurately measuring SMG7 levels, researchers can gain valuable insights into disease mechanisms and potential therapeutic targets.With its easy-to-use protocol and reliable results, the SMG7 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers studying SMG7 biology and its implications in health and disease. Unlock the potential of SMG7 research with this advanced ELISA kit.

Product Name:SMG7 Colorimetric Cell-Based ELISA
Product Code:CBCAB01168
ELISA Type:Cell-Based
Target:SMG7
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The SMG7 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SMG7 protein expression profile in cells. The kit can be used for measuring the relative amounts of SMG7 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on SMG7.

Qualitative determination of SMG7 concentration is achieved by an indirect ELISA format. In essence, SMG7 is captured by SMG7-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: 9887, UniProt ID: Q92540, OMIM: 610964, Unigene: Hs.591463
Gene Symbol:SMG7
Sub Type:None
UniProt Protein Function:Plays a role in nonsense-mediated mRNA decay. Recruits UPF1 to cytoplasmic mRNA decay bodies. Together with SMG5 is thought to provide a link to the mRNA degradation machinery involving exonucleolytic pathways, and to serve as an adapter for UPF1 to protein phosphatase 2A (PP2A), thereby triggering UPF1 dephosphorylation.
NCBI Summary:This gene encodes a protein that is essential for nonsense-mediated mRNA decay (NMD); a process whereby transcripts with premature termination codons are targeted for rapid degradation by a mRNA decay complex. The mRNA decay complex consists, in part, of this protein along with proteins SMG5 and UPF1. The N-terminal domain of this protein is thought to mediate its association with SMG5 or UPF1 while the C-terminal domain interacts with the mRNA decay complex. This protein may therefore couple changes in UPF1 phosphorylation state to the degradation of NMD-candidate transcripts. Alternative splicing results in multiple transcript variants encoding distinct isoforms. [provided by RefSeq, Aug 2011]
UniProt Code:Q92540
NCBI GenInfo Identifier:84028262
NCBI Gene ID:9887
NCBI Accession:Q92540.2
UniProt Secondary Accession:Q92540,Q5T1Q0, Q6PIE0, Q7Z7H9, Q8IXC1, Q8IXC2, B4DRB2 E9PCI0, E9PEH2,
UniProt Related Accession:Q92540
Molecular Weight:127,854 Da
NCBI Full Name:Protein SMG7
NCBI Synonym Full Names:SMG7, nonsense mediated mRNA decay factor
NCBI Official Symbol:SMG7  
NCBI Official Synonym Symbols:EST1C; SGA56M; C1orf16  
NCBI Protein Information:protein SMG7
UniProt Protein Name:Protein SMG7
UniProt Synonym Protein Names:EST1-like protein C; SMG-7 homolog; hSMG-7
Protein Family:Protein
UniProt Gene Name:SMG7  
UniProt Entry Name:SMG7_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-SMG7 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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