Cat. # | Size | Qty. | Price |
---|---|---|---|
12301S | 100 µl |
|
REACTIVITY | H M R Mk |
SENSITIVITY | Endogenous |
MW (kDa) | 100 |
SOURCE | Rabbit |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
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, Rat, Monkey
Hamster, Bovine, Dog, Guinea Pig
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys458 of human APC2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Cell proliferation in all eukaryotic cells depends strictly upon the ubiquitin ligase (E3) activity of the anaphase promoting complex/cyclosome (APC/C), whose main function is to trigger the transition of the cell cycle from metaphase to anaphase. APC/C performs its various functions by promoting the assembly of polyubiquitin chains on substrate proteins, which targets these proteins for degradation by the 26S proteasome (1,2). In humans, twelve different APC/C subunits have been identified. Like all E3 enzymes, APC/C utilizes ubiquitin residues that have been activated by E1 enzymes and then transferred to E2 enzymes. Indeed, APC/C has been shown to interact with UBE2S and UBE2C E2 enzymes, in part, via the RING-finger domain-containing subunit, APC11 (3-5). APC/C activity is also strictly dependent upon its association with multiple cofactors. For example, the related proteins, Cdc20 and Cdh1/FZR1, participate in the recognition of APC/C substrates by interacting with specific recognition elements in these substrates (6), called D-boxes (7) and KEN-boxes (8).
Anaphase-promoting complex subunit 2 (APC2) is a distant member of the cullin family (9,10) that interacts with a RING-H2 finger protein related to Rbx1/Hrt1/Roc1, called APC11, to form the catalytic subcomplex of the APC/C. The APC2/11 subcomplex recruits E2 enzymes such as UBE2C/UBCH10 and is required for the APC/C to catalyze substrate ubiquitination (11). Therefore, APC is a member of the expanding family of cullin-RING finger-based ubiquitin ligases. The physiologic importance of APC2 was underscored by the finding that disruption of murine Apc2 causes embryonic lethality (12).
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