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AF10 Colorimetric Cell-Based ELISA

SKU:
CBCAB01042
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

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AF10 Colorimetric Cell-Based ELISA

The AF10 Colorimetric Cell-Based ELISA Kit offered by Assaygenie is a cutting-edge tool for analyzing angiogenin levels in various biological samples. This innovative kit boasts exceptional sensitivity and specificity, delivering accurate and dependable results for researchers across diverse disciplines.Angiogenin is a key player in angiogenesis, the process of forming new blood vessels. By influencing cell growth and development, angiogenin is implicated in numerous health conditions, including cancer, cardiovascular diseases, and neurodegenerative disorders.

With the AF10 Colorimetric Cell-Based ELISA Kit, scientists can explore the role of angiogenin in these diseases, paving the way for potential therapeutic advancements.Trust Assaygenie's AF10 Colorimetric Cell-Based ELISA Kit to provide precise measurements of angiogenin levels in serum, plasma, and cell culture supernatants. Accelerate your research with this reliable and versatile tool, designed to meet the demands of modern scientific investigation.

Product Name:AF10 Colorimetric Cell-Based ELISA
Product Code:CBCAB01042
ELISA Type:Cell-Based
Target:AF10
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The AF10 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect AF10 protein expression profile in cells. The kit can be used for measuring the relative amounts of AF10 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on AF10.

Qualitative determination of AF10 concentration is achieved by an indirect ELISA format. In essence, AF10 is captured by AF10-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: 8028, UniProt ID: P55197, OMIM: 602409, Unigene: Hs.30385
Gene Symbol:MLLT10
Sub Type:None
UniProt Protein Function:MLLT10: Probably involved in transcriptional regulation. In vitro or as fusion protein with MLL has transactivation activity. Binds to cruciform DNA. A chromosomal aberration involving MLLT10 is associated with acute leukemias. Translocation t(10;11)(p12;q23) with MLL/HRX. The result is a rogue activator protein. A chromosomal aberration involving MLLT10 is associated with diffuse histiocytic lymphomas. Translocation t(10;11)(p13;q14) with PICALM. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Oncoprotein; Transcription factor

Chromosomal Location of Human Ortholog: 10p12

Cellular Component: cytoplasm; nucleoplasm

Molecular Function:protein binding

Biological Process: positive regulation of transcription from RNA polymerase II promoter

Disease: Leukemia, Acute Myeloid

NCBI Summary:This gene encodes a transcription factor and has been identified as a partner gene involved in several chromosomal rearrangements resulting in various leukemias. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2010]
UniProt Code:P55197
NCBI GenInfo Identifier:527504034
NCBI Gene ID:8028
NCBI Accession:P55197.2
UniProt Secondary Accession:P55197,Q5JT37, Q5VX90, Q66K63, B1ANA8,
UniProt Related Accession:P55197
Molecular Weight:20,047 Da
NCBI Full Name:Protein AF-10
NCBI Synonym Full Names:myeloid/lymphoid or mixed-lineage leukemia; translocated to, 10
NCBI Official Symbol:MLLT10  
NCBI Official Synonym Symbols:AF10  
NCBI Protein Information:protein AF-10
UniProt Protein Name:Protein AF-10
UniProt Synonym Protein Names:ALL1-fused gene from chromosome 10 protein
Protein Family:Protein
UniProt Gene Name:MLLT10  
UniProt Entry Name:AF10_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-AF10 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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