Cat. # | Size | Qty. | Price |
---|---|---|---|
31954S | 100 µl |
|
REACTIVITY | M |
SENSITIVITY | Endogenous |
MW (kDa) | 235 |
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 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
Mouse
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val2145 of mouse ABCA7 protein. Antibodies are purified by protein A and peptide affinity chromatography.
ATP-binding cassette (ABC) proteins are membrane-residing transporters that transport substrates across the membrane in an ATP-dependent manner. ABC substrates subject to active transport across the membrane include ions, amino acids, lipids, and sterols (1). ATP-binding cassette sub-family A member 7 (ABCA7) is a member of the ABC family and functions to regulate phospholipid and cholesterol homeostasis in central nervous system (CNS) as well as peripheral tissue. ABCA7, like most ABC transporters, contains two transmembrane domain bundles composed of six membrane-spanning helices and two nucleotide-binding domains. ABCA7 and its closest homolog, ABCA1, are 12A class members of ABCs and both proteins function to transport cholesterol and phospholipids in an apolipoprotein A (apoA) – dependent manner (2,3). ABCA7 is expressed in a variety of tissue and exhibits neuronal and microglial enrichment in the CNS (4). Human genetic studies identified ABCA7 gene variants, including loss-of-function mutations, that associate with late-onset Alzheimer’s disease (AD) (5). ABCA7 dysfunction may contribute directly to AD pathogenesis by accelerating amyloid-β (Aβ) production and/or altering microglia-dependent phagocytosis of the Aβ (4,6,7).
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