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.
MMP13 Antibody recognizes endogenous levels of total MMP13 protein.
Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro275 of human MMP13 protein. Antibodies are purified by protein A and peptide affinity chromatography.
MMP13 (collagenase 3) belongs to the matrix metalloproteinase (MMP) superfamily of enzymes that targets many extracellular proteins, including other proteases, growth factors, cell surface receptors, and adhesion molecules (1, 2). MMP13 is a member of a subgroup of collagenases (including MMP1, MMP8, and MMP18) that play an even more important function targeting fibrillar collagen. MMP13 is synthesized as a latent proenzyme, and proteolytic removal of the inhibitory propeptide domain is required for enzyme activation. MMP13 protein levels are regulated at the transcriptional level, via specific transcription factors and via promoter DNA methylation (3, 4). MMP13 preferentially cleaves Type II collagen, and research studies have shown that aberrant upregulation of MMP13 activity can lead to cartilage loss and osteoarthritis (5, 6). In addition, MMP13 has been shown to promote cancer development, in part through enhancing tumor angiogenesis and metastases (7-9), suggesting that collagenase activity may serve as a useful marker of tumor progression (10).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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