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Tubulin alpha Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00893
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

Tubulin alpha Colorimetric Cell-Based ELISA Kit

The Tubulin Alpha Colorimetric Cell-Based ELISA Kit from AssayGenie is specifically designed for the accurate and sensitive detection of tubulin alpha levels in cell lysates and tissue samples. Tubulin alpha is a key protein involved in cell division, cellular structure, and intracellular transport, making it essential for various cellular processes.This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for researchers studying tubulin alpha in cell biology and cancer research. With its easy-to-use protocol and quick assay time, this kit is ideal for a wide range of applications, allowing for efficient and detailed analysis of tubulin alpha levels in different experimental settings.

Whether investigating cell cycle regulation, microtubule dynamics, or drug screening, the Tubulin Alpha Colorimetric Cell-Based ELISA Kit provides researchers with a valuable tool for understanding the role of tubulin alpha in cellular functions and disease processes. Upgrade your research with this advanced ELISA kit from AssayGenie.

Product Name:Tubulin alpha Colorimetric Cell-Based ELISA
Product Code:CBCAB00893
ELISA Type:Cell-Based
Target:Tubulin alpha
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of Tubulin alpha concentration is achieved by an indirect ELISA format. In essence, Tubulin alpha is captured by Tubulin alpha-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: 7846/10376/84790/113457/7278/112714/7277, UniProt ID: Q71U36/P68363, OMIM: 602529/611603/602530/602528/191110, Unigene: Hs.654422/Hs.713158/Hs.524390/Hs.349695/Hs.503749/Hs.433336/Hs.75318
Gene Symbol:TUBA1A
Sub Type:None
UniProt Protein Function:TUBA1A: Tubulin is the major constituent of microtubules. It binds two moles of GTP, one at an exchangeable site on the beta chain and one at a non-exchangeable site on the alpha-chain. Dimer of alpha and beta chains. Expressed at a high level in fetal brain. Belongs to the tubulin family.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Cytoskeletal

Chromosomal Location of Human Ortholog: 12q13.12

Cellular Component: microtubule; recycling endosome; cytoplasmic microtubule; cytosol; nucleus

Molecular Function:GTPase activity; protein domain specific binding; protein binding; GTP binding; structural constituent of cytoskeleton; structural molecule activity

Biological Process: protein polymerization; 'de novo' posttranslational protein folding; cellular protein metabolic process; protein folding; cell division; organelle organization and biogenesis; cytoskeleton-dependent intracellular transport; mitotic cell cycle; G2/M transition of mitotic cell cycle; microtubule-based process

Disease: Lissencephaly 3

NCBI Summary:Microtubules of the eukaryotic cytoskeleton perform essential and diverse functions and are composed of a heterodimer of alpha and beta tubulins. The genes encoding these microtubule constituents belong to the tubulin superfamily, which is composed of six distinct families. Genes from the alpha, beta and gamma tubulin families are found in all eukaryotes. The alpha and beta tubulins represent the major components of microtubules, while gamma tubulin plays a critical role in the nucleation of microtubule assembly. There are multiple alpha and beta tubulin genes, which are highly conserved among species. This gene encodes alpha tubulin and is highly similar to the mouse and rat Tuba1 genes. Northern blot studies have shown that the gene expression is predominantly found in morphologically differentiated neurologic cells. This gene is one of three alpha-tubulin genes in a cluster on chromosome 12q. Mutations in this gene cause lissencephaly type 3 (LIS3) - a neurological condition characterized by microcephaly, intellectual disability, and early-onset epilepsy caused by defective neuronal migration. Alternative splicing results in multiple transcript variants encoding distinct isoforms. [provided by RefSeq, Jul 2017]
UniProt Code:Q71U36
NCBI GenInfo Identifier:55977864
NCBI Gene ID:7846
NCBI Accession:Q71U36.1
UniProt Secondary Accession:Q71U36,P68363,
UniProt Related Accession:Q71U36
Molecular Weight:~ 55kDa
NCBI Full Name:Tubulin alpha-1A chain
NCBI Synonym Full Names:tubulin alpha 1a
NCBI Official Symbol:TUBA1A  
NCBI Official Synonym Symbols:LIS3; TUBA3; B-ALPHA-1  
NCBI Protein Information:tubulin alpha-1A chain
UniProt Protein Name:Tubulin alpha-1A chain
UniProt Synonym Protein Names:Alpha-tubulin 3; Tubulin B-alpha-1; Tubulin alpha-3 chain
Protein Family:Tubulin
UniProt Gene Name:TUBA1A  
UniProt Entry Name:TBA1A_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-Tubulin alpha 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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