Western blot analysis of extracts from Raw264.7 cells, untreated or LPS-treated (1 μg/ml for 6 h), using iNOS Antibody (Mouse Specific).Learn more about how we get our images.
Western blot analysis of extracts from Raw264.7 cells, untreated or LPS-treated (1 µg/ml for 6 h), using iNOS Antibody (Mouse Specific).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
iNOS Antibody (Mouse Specific) detects endogenous levels of total iNOS protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide sourrounding Ala1130 of mouse iNOS. Antibodies are purified by protein A and peptide affinity chromatography.
Nitric Oxide Synthase (NOS) catalyzes the formation of nitric oxide (NO) and citruline from L-arginine, oxygen and cofactors. Three family members have been characterized: neuronal NOS (nNOS), which is found primarily in neuronal tissue; inducible NOS (iNOS), which is induced by interferon gamma and lipopolysaccharides in the kidney and cardiovascular system; and endothelial NOS (eNOS), which is expressed in blood vessels (1). NO is a messenger molecule with diverse functions throughout the body including the maintenance of vascular integrity, homeostasis, synaptic plasticity, long-term potentiation, learning, and memory (2,3).
Nitric oxide catalyzed by iNOS is involved in host defense against protozoa, bacteria, fungi and viruses. Unlike constitutively expressed eNOS and nNos, iNOS is not usually expressed in quiescent cells. iNOS is transcriptionally induced in response to bacterial endotoxins such as LPS and proinflammatory cytokines in macrophages and various other cell types. Transcription factors involved in iNOS transcription include NF-κB, AP-1 and STAT. Different signaling pathways either promote (Jak1/2, PKC, c-Raf, p38 MAP kinase and p44/42 MAP kinase) or inhibit (PI3 kinase) iNOS expression depending on stimulus and cell type (4).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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