Cat. # | Size | Qty. | Price |
---|---|---|---|
97647S | 100 µl |
|
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | 76 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
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 utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.
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.
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: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 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) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.
When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.
posted December 2008
revised October 2021
Protocol Id: 409
Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly375 of human PADI2 protein.
Peptidyl arginine deiminase (PAD) proteins are a family of Ca2+-dependent enzymes that catalyze the post-translational conversion of arginine to citrulline. There are currently five known PAD isozymes in humans, referred to as PADI1-4 and PADI6 (1). Among these isozymes, peptidyl arginine deiminase type 2 (PADI2) is the most widely expressed, being found in skeletal muscle, brain, colon, breast, macrophages, spleen, and spinal cord tissue, among others (1,2). In normal mouse development, PADI2 expression levels are elevated from 18 days to 2 months of age, and gradually decrease from 3 months onward (3). Some of the most well studied PADI2 substrates include vimentin, actin, myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and histones (4). PADI2-mediated citrullination has been shown to be involved in neurodegeneration and inflammatory response-associated diseases such as multiple sclerosis (MS), Alzheimer’s disease (AD), psoriasis, and rheumatoid arthritis (5). Excessive PAD-mediated deimination of MBP is believed to be a major contributor to MS disease progression, while elevated levels of citrullinated GFAP and vimentin proteins have been found in the brains of AD patients (2,4). PADI2 has also been found to play a role in the progression of several types of cancers, including colorectal, breast, and prostate (5-7).
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