Guanine Nucleotide Binding Protein (G Protein), beta Polypeptide 2-Like 1 (GNB2L1) (AA 113-317) antibody

Antigen: Guanine Nucleotide Binding Protein (G Protein), beta Polypeptide 2-Like 1 (GNB2L1)

Synonyms: MGC53289, C12-3, GNB2L1

Binding Site: AA 113-317

Clonality: Monoclonal (20)

Host: Mouse

Reactivity: Rat (Rattus)

Conjugate: Un-conjugated

Application: Western Blotting (WB), BioImaging (BI), Immunohistochemistry (IHC), Immunoprecipitation (IP)

Catalog no.: ABIN967797

Additional Information

Alternative name: RACK1

Immunogen: Rat RACK1 aa 113-317

Cross-Reactivity: Human, Cow (Bovine), Chicken, Dog (Canine), Frog, Mouse (Murine)

Format: Liquid

Isotype: IgM

Clone: 20

Description: Several proteins which specifically bind to PKC have been classified as RACKs (receptors for activated C-kinase). RACK1 was cloned from a rat brain cDNA expression library by screening for proteins that bind PKC in the presence of phosphatidylserine, diacylglycerol, and calcium in a PKC overlay assay. By sequence homology, RACK1 appears to belong to a superfamily that includes the ß subunit of G proteins. All of these proteins contain five to eight internal repeat elements known as WD40 motifs, which appear to have a role in protein-protein interactions. In addition, RACK1 contains two short sequences homologous to a PKC-binding sequence identified in Annexin I and in the brain PKC inhibitor KCIP. The binding of RACK1 to PKC is dose-dependent and occurs at a site on PKC that is distinct from the catalytic domain, indicating that RACK1 is not a PKC substrate.

Characteristics: 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to us for technical protocols.
3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
6. Triton is a trademark of the Dow Chemical Company.

Molecular Weight: 36 kDa

Comments:

Related Products: ABIN968537

Application Details

Application Notes: Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in an 96-well Imaging Plate and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 myl of Fixation Buffer to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either 90% methanol, or Triton™ X-100: a. Add 100 myl of -20°C 90% methanol to each well and incubate for 5 minutes at RT. OR b. Add 100 myl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 myl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 myl of to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 myl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 myl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 myl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 myl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 myl per well of 2 myg/ml Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.

Concentration: 250 µg/ml

Purity: Purified

Purification: Purified from tissue culture supernatant or ascites by affinity chromatography.

Buffer: Aqueous buffered solution containing BSA, glycerol.

Preservative: 0.09% Sodium azide.

Storage: Store undiluted at -20° C.

Research Area: Kinases/Phosphatases

Restrictions: For Research Use only

Images

Western blot analysis of RACK1 on a Jurkat lysate. Lane 1: 1:2500, lane 2: 1:5000, lane 3: 1:10000 dilution of the RACK1 antibody.

Immunofluorescent staining of A549 (ATCC CCL-185) cells. Cells were seeded in a 96 well imaging plate at ~ 10 000 cells per well. After overnight incubation, cells were stained using the alcohol perm protocol and the anti-RACK1 antibody. The second step reagent was FITC goat anti mouse Ig. The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective. This antibody also stained U-2 OS (ATCC HTB-96) and HeLa (ATCC CCL-2) cells using both the Triton™ X-100 and alcohol perm protocols.

anti-Guanine Nucleotide Binding Protein (G Protein), beta Polypeptide 2-Like 1 (GNB2L1) (AA 113-317) antibody (Image 3)

Publications

Product: Smart, Ying, Anderson: "Hormonal regulation of caveolae internalization." in: The Journal of cell biology, Vol. 131, Issue 4, pp. 929-38, 1996 (PubMed).

Ron, Chen, Caldwell et al.: "Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, Issue 3, pp. 839-43, 1994 (PubMed).

Chang, Chiang, Cartwright: "The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1." in: The Journal of biological chemistry, Vol. 276, Issue 23, pp. 20346-56, 2001 (PubMed).

Liedtke, Yun, Kyle et al.: "Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor." in: The Journal of biological chemistry, Vol. 277, Issue 25, pp. 22925-33, 2002 (PubMed).

Birikh, Sklan, Shoham et al.: "Interaction of "readthrough" acetylcholinesterase with RACK1 and PKCbeta II correlates with intensified fear-induced conflict behavior." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, Issue 1, pp. 283-8, 2003 (PubMed).