Cat. # | Size | Qty. | Price |
---|---|---|---|
12304S | 100 µl |
|
REACTIVITY | H M Mk |
SENSITIVITY | Endogenous |
MW (kDa) | 78, 82 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2021
Protocol Id: 410
Human, Mouse, Monkey
Rat, Hamster, Bovine, Dog, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys485 of human CIN85 protein.
CIN85 was independently identified as Cbl-interacting protein of 85 kDa (1), Ruk (regulator of ubiquitous kinase) (2), SETA (SH3 domain-containing gene expressed in tumorigenic astrocytes) (3), and SH3KBP1 (SH3 domain kinase binding protein 1) (4). The genes encoding these proteins were isolated from either human (CIN85), rat (Ruk and SETA), or mouse (SH3KBP1) sources and share between 92% and 97% sequence identity, suggesting that they represent homologues of one gene. Differential promoter usage and alternative splicing is thought to occur in a tissue specific and developmentally regulated manner to generate a complex expression pattern of various transcripts and encoded protein isoforms (5). The main isoform in humans, CIN85, contains three N-terminal SH3 domains, a proline-rich region harboring several P-X-X-P motifs that provide recognition sites for SH3 domain-containing proteins, a PEST sequence implicated in CIN85 degradation, and a C-terminal coiled-coil region for oligomerization (1,2,5,6). The other molecular variants of CIN85 are shorter, N-terminally truncated proteins lacking one, two, or all three of the SH3 domains (1,5,6-8). Proteomic screens suggest that CIN85 is phosphorylated at multiple sites and the role of phosphorylation of some of these sites in regulation of intra- and intermolecular interactions of CIN85 cannot be excluded. CIN85 belongs to the CD2AP/CMS family of adaptor proteins and has been shown to interact with signaling molecules such as c-Cbl, Cbl-b, BLNK, p85/PI3K, GRB2, p130 Cas, and endophilins to coordinate the activity of multiple signaling cascades. Indeed, a growing body of evidence suggests that CIN85 is required for the regulation of a variety of cellular processes including vesicle-mediated transport (9-12), signal transduction (13,14), and cytoskeleton remodelling (15).
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