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UBE1L Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00896
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Signal Transduction
Reactivity:
Human
Detection Method:
Colorimetric
€599
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Description

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UBE1L Colorimetric Cell-Based ELISA Kit

The UBE1L Colorimetric Cell-Based ELISA Kit is a powerful tool for the detection of UBE1L levels in cell lysates and tissue samples. This kit offers exceptional sensitivity and specificity, allowing for precise and reliable results in various research applications.UBE1L, also known as Ubiquitin-Activating Enzyme E1-like protein, is a key enzyme in the ubiquitination pathway, playing a critical role in protein degradation and regulation. Dysregulation of UBE1L has been implicated in various diseases, including cancer, neurodegenerative disorders, and autoimmune diseases, making it a valuable target for research and drug development.

By accurately measuring UBE1L levels, researchers can gain valuable insights into the molecular mechanisms underlying these diseases and potentially identify new therapeutic targets. The UBE1L Colorimetric Cell-Based ELISA Kit offers a simple and efficient method for studying UBE1L expression, making it an indispensable tool for researchers in the field of molecular biology and drug discovery.

Product Name:UBE1L Colorimetric Cell-Based ELISA
Product Code:CBCAB00896
ELISA Type:Cell-Based
Target:UBE1L
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The UBE1L Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect UBE1L protein expression profile in cells. The kit can be used for measuring the relative amounts of UBE1L in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on UBE1L.

Qualitative determination of UBE1L concentration is achieved by an indirect ELISA format. In essence, UBE1L is captured by UBE1L-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: 7318, UniProt ID: P41226, OMIM: 191325, Unigene: Hs.16695
Gene Symbol:UBA7
Sub Type:None
UniProt Protein Function:UBE1L: Activates ubiquitin by first adenylating with ATP its C- terminal glycine residue and thereafter linking this residue to the side chain of a cysteine residue in E1, yielding an ubiquitin- E1 thioester and free AMP. Belongs to the ubiquitin-activating E1 family.
UniProt Protein Details:

Protein type:Ubiquitin conjugating system; Ubiquitin ligase; EC 6.3.2.19

Chromosomal Location of Human Ortholog: 3p21

Cellular Component: cytosol; nucleoplasm; nucleus

Molecular Function:ISG15 activating enzyme activity; protein binding; ubiquitin activating enzyme activity; ubiquitin-protein ligase activity

Biological Process: bypass DNA synthesis; ISG15-protein conjugation; modification-dependent protein catabolic process; negative regulation of interferon type I production; protein modification process

NCBI Summary:The modification of proteins with ubiquitin is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation. Ubiquitination involves at least three classes of enzymes: ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s. This gene encodes a member of the E1 ubiquitin-activating enzyme family. The encoded enzyme is a retinoid target that triggers promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARalpha) degradation and apoptosis in acute promyelocytic leukemia, where it is involved in the conjugation of the ubiquitin-like interferon-stimulated gene 15 protein. [provided by RefSeq, Jul 2008]
UniProt Code:P41226
NCBI GenInfo Identifier:215273977
NCBI Gene ID:7318
NCBI Accession:P41226.2
UniProt Secondary Accession:P41226,Q9BRB2,
UniProt Related Accession:P41226
Molecular Weight:111,694 Da
NCBI Full Name:Ubiquitin-like modifier-activating enzyme 7
NCBI Synonym Full Names:ubiquitin like modifier activating enzyme 7
NCBI Official Symbol:UBA7  
NCBI Official Synonym Symbols:D8; UBE2; UBA1B; UBE1L  
NCBI Protein Information:ubiquitin-like modifier-activating enzyme 7
UniProt Protein Name:Ubiquitin-like modifier-activating enzyme 7
UniProt Synonym Protein Names:D8; Ubiquitin-activating enzyme E1 homolog
Protein Family:Ubiquitin-like modifier-activating enzyme
UniProt Gene Name:UBA7  
UniProt Entry Name:UBA7_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-UBE1L 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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