Western blot analysis of extracts from various cell lines using Phospho-LDHA (Tyr10) Antibody.
Western blot analysis of extracts from 293T cells transfected with a construct expressing DYKDDDDK-tagged wild-type (WT) full-length human LDHA (WT hLDHA-DDK) alone or in conjuction with full-length human FGFR1 (hFGFR1), using Phospho-LDHA (Tyr10) Antibody (upper) or DYKDDDDK Tag (9A3) Mouse mAb #8146 (lower). The DYKDDDDK-tagged WT hLDHA expression construct was kindly provided by Dr. Jing Chen at Emory University School of Medicine.
|REACTIVITY||H M R|
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
Phospho-LDHA (Tyr10) Antibody recognizes endogenous levels of LDHA protein only when phosphorylated at Tyr10.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr10 of human LDHA protein. Antibodies are purified by protein A and peptide affinity chromatography.
Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and NADH to lactate and NAD+. When the oxygen supply is too low for mitochondrial ATP production, this reaction recycles NADH generated in glycolysis to NAD+, which reenters glycolysis. The major form of LDH found in muscle cells is the A (LDHA) isozyme. The LDHA promoter contains HIF-1α binding sites (1). LDHA expression is induced under hypoxic conditions (2). During intensive and prolonged muscle exercise, lactate accumulates in muscle cells when the supply of oxygen does not meet demand. When oxygen levels return to normal, LDH converts lactate to pyruvate to generate ATP in the mitochondrial electron transport chain.
Recent studies indicate that LDHA is phosphorylated at Tyr10 and Tyr83 by the receptor tyrosine kinase FGFR1 (3). Phosphorylation at Tyr10 up-regulates the activity of LDHA and is found in a variety of human cancer cells (3). Results further suggest that tyrosine phosphorylation regulates the NADH/NAD+ redox homeostasis and thus promotes the Warburg effect and tumor cell growth (3).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.