Western blot analysis of extracts from PANC-1 cells or HeLa cells, vehicle-treated (-) or treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (100 μM, 2 hr; +), using PGAM5 (Long Isoform) Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
|REACTIVITY||H M R Mk|
|MW (kDa)||28, 30|
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.
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
PGAM5 (Long Isoform) Antibody recognizes endogenous levels of total PGAM5 long isoform protein. This antibody specifically recognizes the PGAM5 long isoform (UniProt #Q96HS1-1) and does not recognize the PGAM5 short isoform (UniProt #Q96HS1-2).Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human PGAM5 long isoform (UniProt #Q96HS1-1). Antibodies are purified by protein A and peptide affinity chromatography.
Phosphoglycerate mutase family 5 (PGAM5) is a mitochondrial serine/threonine protein phosphatase (1,2). PGAM5 is required for necrosis induced by TNF-α or reactive oxygen species (2). In addition, PGAM5 dephosphorylates FUNDC1 to induce mitophagy under mitochondrial stresses (3). PGAM5 is also essential for RIPK1-mediated mitophagy activation during extracellular matrix (ECM) detachment of cancer cells (4). Furthermore, PGAM5 is cleaved by the protease presenilin-associated rhomboid-like protein (PARL) in response to mitochondrial stresses (such as induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment). The cleaved PGAM5, released from the mitochondrial membrane, dephosphorylates β-catenin in the cytosol leading to its stabilization. Increased levels of β-catenin stimulate mitochondrial biogenesis (5).
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