Cat. # | Size | Qty. | Price |
---|---|---|---|
14581S | 100 µl |
|
REACTIVITY | M R |
SENSITIVITY | Endogenous |
MW (kDa) | 160-200 |
SOURCE | Rabbit |
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
Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg77 of mouse NKCC1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
The electroneutral cation-chloride-coupled co-transporter (SLC12) gene family comprises bumetanide-sensitive Na+/K+/Cl- (NKCC), thiazide-sensitive Na+/Cl-, and K+/Cl- (KCC) co-transporters. SLC12A1/NKCC2 and SLC12A2/NKCC1 regulate cell volume and maintain cellular homeostasis in response to osmotic and oxidative stress (1). The broadly expressed NKCC1 is thought to play roles in fluid secretion (i.e. salivary gland function), salt balance (i.e. maintenance of renin and aldosterone levels), and neuronal development and signaling (2-7). During neuronal development, NKCC1 and KCC2 maintain a fine balance between chloride influx (NKCC1) and efflux (KCC2), which regulates γ-aminobutyric acid (GABA)-mediated neurotransmission (3). Increased NKCC1 expression in immature neurons maintains high intracellular chloride levels that result in inhibitory GABAergic signaling; KCC2 maintains low intracellular chloride levels and excitatory GABAergic responses in mature neurons (4,5,8). Deletion of NKCC1 impairs NGF-mediated neurite outgrowth in PC-12D cells while inhibition of NKCC1 with bumetanide inhibits re-growth of axotomized dorsal root ganglion cells (6,7). Defective chloride homeostasis in neurons is linked to seizure disorders that are ameliorated by butemanide treatment, indicating that abnormal NKCC1 and NKCC2 expression or signaling may play a role in neonatal and adult seizures (9-12). NKCC1 is found as a homodimer or within heterooligomers with other SLC12 family members. This transport protein associates with a number of oxidative- and osmotic-responsive kinases that bind, phosphorylate, and activate NKCC1 co-transporter activity (13-16). In response to decreased intracellular chloride concentrations, Ste20-related proline-alanine-rich kinase (SPAK) phosphorylates NKCC1 to increase co-transporter activity and promote chloride influx (16-19). Oxidative stress response kinase 1 (OSR1) also phosphorylates and activates NKCC1 in response to oxidative stress (14).
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