GAK antibody (AA 1-1305)

Catalog No. ABIN5542247

This Mouse Monoclonal antibody specifically detects GAK in WB. It exhibits reactivity toward Human.

Quick Overview for GAK antibody (AA 1-1305) (ABIN5542247)

Target: GAK (Cyclin G Associated Kinase (GAK))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This GAK antibody is un-conjugated

Application: Western Blotting (WB)

Clone: 1C2

Product Details anti-GAK Antibody

Binding Specificity: AA 1-1305

Specificity: This antibody reacts with GAK.

Purification: Protein A agarose

Immunogen: Recombinant full-length rat GAK (1-1305 aa)

Isotype: IgG1

Application Details

Application Notes: Western blot: 1 μg/mL for chemiluminescence detection system. For details see protocol below.

Protocol: SDS-PAGE & Western Blotting 1) Wash the 1x10 7 cells 3 times with PBS and suspend with 1 mL of Laemmli's sample buffer. 2) Boil the samples for 2 minutes and centrifuge. Load 20 μ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 3) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20 % MeOH). See the manufacture's manual for precise transfer procedure. 4) To reduce nonspecific binding, soak the membrane in 5 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 5) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1 % skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.) 6) Wash the membrane with PBS-T [0.05 % Tween-20 in PBS] (5 minutes x 3 times). 7) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG diluted with 1 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 8) Wash the membrane with PBS-T (5 minutes x 3 times). 9) Wipe excess buffer on the membrane, then incubate it with appropriate chemilumin escence reagent for 1 minute. Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 10) Expose to an X-ray film in a dark room for 10 minutes. Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting HeLa, HL-60, L5178Y)

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. No preservative is contained.

Preservative: Azide free

Storage: -20 °C

Storage Comment: Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Target Details for GAK

Target: GAK (Cyclin G Associated Kinase (GAK))

Alternative Name: gak

Background: He cell cycle of eukaryotes is regulated by cyclins and cyclin-dependent protein kinases (CDKs). Cyclin G1 exhibits closest sequence identity to the cyclin A family. Identification of cyclin G1 as a direct transcriptional target of the p53 tumor suppressor gene product indicates that cyclin G functions downstream of p53. GAK (a cyclin G associated kinase), an association partner of cyclin G1 and CDK5, which is expressed ubiquitously, with the highest level of expression being observed in the testis. The predicted amino acid sequence of GAK possesses an N-terminal Ser/Thr protein kinase catalytic domain with a long C-terminal extension. There are two putative dinucleotide-binding motifs in the kinase domain suggesting that GAK activity is some how modulated by dinucleotides such as ADP or GDP. As GAK lacks PSTAIR motif, it can not be considered a member of the CDK family. Kinase activities of GAK is almost throughout the cell cycle. In the middle part of the molecule, GAK resembles both tensin, an actin-binding component of adhesion plaques (also called focal contacts) and other submembranous cyto skeletal structure, and auxilin, a coat component of brain clathrin-coated vesicles. The domain homologous to tensin and auxilin occupies more than 50 % of the whole molecule. A leucine zipper motif, whose physiological meaning is unknown, also located in this domain. A phosphorylation recognition site for a tyrosine kinase is located at position 403-410 (KGDLDISY). One, 19 and 24 potential phosphorylation sites for cAMP-dependent protein kinase, protein kinase C and casein kinase II protein kinase, respectively, are also located through whole mol ecule. The presence of a Ser/Thr kinase domain at the N-terminal portion of the molecule suggests that GAK plays a role as a modulator of signal transduction, by relaying the phosphorylation signal from an unidentified tyrosine kinase to unidentified GAK phosphorylation targets.

UniProt: O14976