Western blot analysis of extracts from RAW 264.7 cells, untreated (-) or treated with LPS #14011 (1 μg/ml, overnight; +), using MCP-1 (E9R7Z) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
|MW (kDa)||11-13, 20-30|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
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
MCP-1 (E9R7Z) Rabbit mAb recognizes endogenous levels of total MCP-1 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Cys75 of mouse MCP-1 protein.
Monocyte chemotactic protein-1 (MCP-1), also known as CCL2, monocyte chemotactic activating factor (MCAF) or glioma-derived chemotactic factor-2 (GDCF-2), is the product of the human JE gene and a member of the family of C-C (or β) chemokines (1-4). The predicted molecular weight of MCP-1 protein is 11-13 kDa, but it may migrate at 20-30 kDa due to glycosylation. MCP-1 is secreted by a variety of cell types in response to pro-inflammatory stimuli and was originally described for its chemotactic activity on monocytes. This activity has led to studies demonstrating its role in diseases characterized by monocyte infiltrates such as psoriasis (5), rheumatoid arthritis (6) and atherosclerosis (7). MCP-1 may also contribute to tumor progression and angiogenesis (8). Signaling by MCP-1 is mediated by the G-protein coupled receptor CCR2 (9).
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