REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H M R | Endogenous | 46, 61 | Rabbit IgG |
Western blot analysis of extracts from rat brain and mouse brain using Non-phospho-STEP (Ser221) (D74H3) XP® Rabbit mAb. The nonphospho-specificity of the antibody was verified by preincubating the antibody without peptide, with STEP61 (Ser221) non-phosphopeptide, or with STEP61 (Ser221) phosphopeptide prior to incubation with the membrane.
Learn more about how we get our images.Confocal immunofluorescent analysis of rat hippocampus (left) and striatum (right) using Non-phospho-STEP (Ser221) (D74H3) XP® Rabbit mAb (green) and α/β-Synuclein (Syn205) Mouse mAb #2644 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Learn more about how we get our images.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: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2018
Protocol Id: 410
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
Cover sections with 4% formaldehyde dilute in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised July 2016
Protocol Id: 151
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
Immunofluorescence (Frozen) | 1:50 |
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Non-phospho-STEP (Ser221) (D74H3) XP® Rabbit mAb detects endogenous levels of STEP61 protein only when dephosphorylated at Ser221 and of STEP46 protein when dephosphorylated at Ser49.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic nonphosphopeptide corresponding to residues surrounding Ser221 of human STEP61 protein.
Striatal enriched phosphatase (STEP, also known as PTPN5), is a protein tyrosine phosphatase expressed in dopaminoceptive neurons of the central nervous system (1). Alternative splicing produces the cytosolic STEP46 and the membrane-associated STEP61 isoforms of STEP. Dopamine activates D1 receptors and PKA, which in turn phosphorylate both isoforms of STEP. Phosphorylation of STEP61 occurs at Ser160 and Ser221, while STEP46 is phosphorylated at Ser49 (equivalent to Ser221 of STEP61) (2). NMDA-mediated activation of STEP is an important mechanism for regulation of Erk activity in neurons (3). Furthermore, STEP is involved in the regulation of both NMDAR and AMPAR trafficking (4,5). Due to its importance in cognitive function, STEP may play a role in Alzheimer's disease (1).
Activity of STEP61 is reduced upon phosphorylation of Ser221 (Ser49 of STEP46) due to lower affinity for its substrates (2).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. DRAQ5 is a registered trademark of Biostatus Limited.
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Product # | Size | Price |
---|---|---|
5659S | 100 µl | $ 336.0 |