ABL1 (Phospho-Tyr245) Fluorometric Cell-Based ELISA Kit

Description

Description

ABL1 (Phospho-Tyr245) Cell-Based ELISA Kit

The ABL1 (Phospho-Tyr245) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor ABL1 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ABL1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ABL1 phosphorylation.

How does our ABL1 (Phospho-Tyr245) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of ABL1 (Phospho-Tyr245) concentration is achieved by an indirect ELISA format. In essence, ABL1 (Phospho-Tyr245) is captured by ABL1 (Phospho-Tyr245)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. 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 RFU values.
2.	An antibody against the nonphosphorylated counterpart of ABL1 (Phospho-Tyr245) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated ABL1 can be used to normalize the RFU value for phosphorylated ABL1.

ABL1 (Phospho-Tyr245) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	ABL1 (Phospho-Tyr245) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00038
Description:	The ABL1 (Phospho-Tyr245) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor ABL1 (Phospho-Tyr245) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ABL1 (Phospho-Tyr245) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on ABL1 phosphorylation.
Dynamic Range:	> 5000 Cells
Detection Method:	Fluorometric
Storage/Stability:	4°C/6 Months
Reactivity:	Human, Mouse, Rat
Assay Type:	Cell-Based ELISA
Database Links:	Gene ID: 25, UniProt ID: P00519, OMIM #: 189980/608232, Unigene #: Hs.431048
Format:	Two 96-Well Plates
NCBI Gene Symbol:	ABL1
Sub Type:	Phospho
Target Name:	Phospho-ABL1 (Tyr245)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components	Quantity
96-Well Black Cell CultureClear-Bottom Microplate	2 plates
10X TBS	24 ml
Quenching Buffer	24 ml
Blocking Buffer	50 ml
15X Wash Buffer	50 ml
Primary Antibody Diluent	12 ml
100x Anti-Phospho Target Antibody	60 µl
100x Anti-Target Antibody	60 µl
Anti-GAPDH Antibody	110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody	6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody	6 ml
Adhesive Plate Seals	2 seals

Additional equipment and materials required

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

Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
Micropipettes capable of measuring volumes from 1 µl to 1 ml
Deionized or sterile water (ddH2O)
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)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.	Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. 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 3 minutes. 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 3 minutes each time.
9.	Dispense 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 3 minutes each time.
11.	Add 50 µl of Primary Antibody Mixture P to corresponding wells for ABL1 (Phospho-Tyr245) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total ABL1 detection. Cover the plate 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 3 minutes each time.
13.	Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.	Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.	Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

ABL1 (Phospho-Tyr245) - Protein Information

UniProt Protein Function:	Non-receptor tyrosine-protein kinase that plays a role in many key processes linked to cell growth and survival such as cytoskeleton remodeling in response to extracellular stimuli, cell motility and adhesion, receptor endocytosis, autophagy, DNA damage response and apoptosis. Coordinates actin remodeling through tyrosine phosphorylation of proteins controlling cytoskeleton dynamics like WASF3 (involved in branch formation); ANXA1 (involved in membrane anchoring); DBN1, DBNL, CTTN, RAPH1 and ENAH (involved in signaling); or MAPT and PXN (microtubule-binding proteins). Phosphorylation of WASF3 is critical for the stimulation of lamellipodia formation and cell migration. Involved in the regulation of cell adhesion and motility through phosphorylation of key regulators of these processes such as BCAR1, CRK, CRKL, DOK1, EFS or NEDD9. Phosphorylates multiple receptor tyrosine kinases and more particularly promotes endocytosis of EGFR, facilitates the formation of neuromuscular synapses through MUSK, inhibits PDGFRB-mediated chemotaxis and modulates the endocytosis of activated B-cell receptor complexes. Other substrates which are involved in endocytosis regulation are the caveolin (CAV1) and RIN1. Moreover, ABL1 regulates the CBL family of ubiquitin ligases that drive receptor down-regulation and actin remodeling. Phosphorylation of CBL leads to increased EGFR stability. Involved in late-stage autophagy by regulating positively the trafficking and function of lysosomal components. ABL1 targets to mitochondria in response to oxidative stress and thereby mediates mitochondrial dysfunction and cell death. ABL1 is also translocated in the nucleus where it has DNA-binding activity and is involved in DNA-damage response and apoptosis. Many substrates are known mediators of DNA repair: DDB1, DDB2, ERCC3, ERCC6, RAD9A, RAD51, RAD52 or WRN. Activates the proapoptotic pathway when the DNA damage is too severe to be repaired. Phosphorylates TP73, a primary regulator for this type of damage-induced apoptosis. Phosphorylates the caspase CASP9 on 'Tyr-153' and regulates its processing in the apoptotic response to DNA damage. Phosphorylates PSMA7 that leads to an inhibition of proteasomal activity and cell cycle transition blocks. ABL1 acts also as a regulator of multiple pathological signaling cascades during infection. Several known tyrosine-phosphorylated microbial proteins have been identified as ABL1 substrates. This is the case of A36R of Vaccinia virus, Tir (translocated intimin receptor) of pathogenic E.coli and possibly Citrobacter, CagA (cytotoxin-associated gene A) of H.pylori, or AnkA (ankyrin repeat-containing protein A) of A.phagocytophilum. Pathogens can highjack ABL1 kinase signaling to reorganize the host actin cytoskeleton for multiple purposes, like facilitating intracellular movement and host cell exit. Finally, functions as its own regulator through autocatalytic activity as well as through phosphorylation of its inhibitor, ABI1.
NCBI Summary:	This gene is a protooncogene that encodes a protein tyrosine kinase involved in a variety of cellular processes, including cell division, adhesion, differentiation, and response to stress. The activity of the protein is negatively regulated by its SH3 domain, whereby deletion of the region encoding this domain results in an oncogene. The ubiquitously expressed protein has DNA-binding activity that is regulated by CDC2-mediated phosphorylation, suggesting a cell cycle function. This gene has been found fused to a variety of translocation partner genes in various leukemias, most notably the t(9;22) translocation that results in a fusion with the 5' end of the breakpoint cluster region gene (BCR; MIM:151410). Alternative splicing of this gene results in two transcript variants, which contain alternative first exons that are spliced to the remaining common exons. [provided by RefSeq, Aug 2014]
UniProt Code:	P00519
NCBI GenInfo Identifier:	85681908
NCBI Gene ID:	25
NCBI Accession:	P00519.4
UniProt Secondary Accession:	P00519,Q13869, Q13870, Q16133, Q17R61, Q45F09, A3KFJ3
UniProt Related Accession:	P00519
Molecular Weight:	124,955 Da
NCBI Full Name:	Tyrosine-protein kinase ABL1
NCBI Synonym Full Names:	ABL proto-oncogene 1, non-receptor tyrosine kinase
NCBI Official Symbol:	ABL1
NCBI Official Synonym Symbols:	ABL; JTK7; p150; c-ABL; v-abl; c-ABL1; bcr/abl
NCBI Protein Information:	tyrosine-protein kinase ABL1
UniProt Protein Name:	Tyrosine-protein kinase ABL1
UniProt Synonym Protein Names:	Abelson murine leukemia viral oncogene homolog 1; Abelson tyrosine-protein kinase 1; Proto-oncogene c-Abl; p150
Protein Family:	Tyrosine-protein kinase
UniProt Gene Name:	ABL1
UniProt Entry Name:	ABL1_HUMAN