Western blot analysis of STEP was performed by loading 25ug of Wild Type (WT, right lane) and STEP Knockout (KO, left lane) rat striatum lysates, and 10ul of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for 1 hour at room temperature. STEP was detected at ~61kD and ~46kD in WT sample using a STEP monoclonal antibody (Product # MA1-158) at a concentration of 1ug/ml overnight at 4C on a rocking platform, followed by a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:10,000 for at least 1 hour at room temperature. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34076).
|Tested species reactivity||Rat|
|Host / Isotype||Mouse / IgG1|
|Immunogen||A synthetic peptide of STEP46 (amino acids 57-74: MCTPGCNEEGFGYLVSPR)|
|Storage buffer||PBS, pH 7.2, with 30% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Immunoprecipitation (IP)||2 µg|
|Western Blot (WB)||1:200-1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
By Western blot, MA1-158 detects both STEP 46 and STEP 61. By IP, STEP61 was preferentially pulled down, along with a non-specific protein at ~50kDa.
Rat brain lysate can be used as positive control.
Tyrosine phosphorylation plays a critical role in many physiological processes. There is a fine balance between phosphorylation caused by protein tyrosine kinases and actions of protein tyrosine phosphatases, that regulates these physiological processes. Striatal enriched phosphatase (STEP) is part of a family of protein tyrosine phosphatases enriched within the CNS and is a component of the NMDAR complex. It is expressed within dopaminoceptive neurons in the CNS and particularly enriched within the basal ganglia and related structures. STEP61 is a membrane-associated isoform and STEP46 is a cytosolic protein. In response to transient hypoxia-ischemia in the brain, STEP61 is proteolytically cleaved by calpain, thereby releasing a smaller isoform, STEP33.
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