Western blot analysis of extracts from 293 cells transfected with either wild-type or threonine to alanine mutations at the respective phosphorylation sites of the MARK family members, using Phospho-MARK Family (Activation Loop) Antibody. Transfected constructs contain a GST and HA tag. Transfection efficiency monitored with HA Antibody, #2367.
Western blot analysis of extracts from Raji and BaF3 cells, showing endogenous levels of phosphorylated MARK family members, using Phospho-MARK Family (Activation Loop) Antibody.
|REACTIVITY||H M R|
|MW (kDa)||80 to 95|
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-MARK Family (Activation Loop) Antibody detects endogenous levels of phosphorylated MARK family members, MARK1 at threonine 215, MARK2 at threonine 208, and MARK3 at threonine 234 (A.K.A. 211 in isoforms 3-6) . This antibody does not react with MARK4.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding threonine 215 of human MARK1. Antibodies were purified by protein A and peptide affinity chromatography.
Microtubule associated proteins regulate the stability of microtubules and control processes such as cell polarity/differentiation, neurite outgrowth, cell division and organelle trafficking (1). The MARK (MAP/microtubule affinity-regulating kinases) family (MARK1-4) of serine/threonine kinases was identified based on their ability to phosphorylate microtubule-associated proteins (MAPs) including tau, MAP2 and MAP4 (2-6). MARK proteins phosphorylate MAPs within their microtubule binding domains, causing dissociation of MAPs from microtubules and increased microtubule dynamics (2-4). In the case of tau, phosphorylation has been hypothesized to contribute to the formation of neurofibrillary tangles observed in Alzheimer's disease. Overexpression of MARK leads to hyperphosphorylation of MAPs, morphological changes and cell death (4). The tumor suppressor kinase LKB1 phosphorylates MARK and the closely related AMP-kinases within their T-loops, leading to increased activity (7).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.