Cat. # | Size | Qty. | Price |
---|---|---|---|
14547S | 100 µl |
|
REACTIVITY | M |
SENSITIVITY | Endogenous |
MW (kDa) | 15 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Immunoprecipitation | 1:50 |
Immunofluorescence (Frozen) | 1:100 |
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
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
Cover sections with 4% formaldehyde dilute in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised July 2016
Protocol Id: 151
Mouse
Human
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to full-length human CART protein.
Cocaine- and amphetamine-regulated transcript (CART) peptides are neurotransmitters of 39 and 47 amino acids that are involved in a variety of physiological processes. The CART precursor, a polypeptide of 116 residues, requires prohormone/proprotein convertase-mediated endoproteolytic cleavage to produce the two active peptides (1). CART peptides are found in several neuroendocrine tissues such as the brain, pituitary, adrenals, and pancreas (2). Hypothalamic CART is regulated by leptin, and plays a role in appetite and feeding behavior (3). Mesolimbic CART is regulated by CREB and may play a role in drug abuse behaviors by mediating some of CREB effects (4). Pancreatic CART is found in islet endocrine cells and parasympathetic and sensory nerves. It inhibits glucose-stimulated insulin secretion and has been found to be up-regulated in beta cells in animal model of diabetes (5). A missense mutation in the corresponding CART gene can correlate with susceptibility to obesity and reduced resting energy expenditure (6).
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