REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H M R | Endogenous | 18 | Rabbit IgG |
Western blot analysis of extracts from mouse brain, untreated (-) or λ phosphatase-treated (+), using Phospho-α-Synuclein (Ser129) (D1R1R) Rabbit mAb (upper) or α-Synuclein (D37A6) XP® Rabbit mAb #4179 (lower).
Learn more about how we get our imagesConfocal immunofluorescent analysis of mouse hippocampus using Phospho-α-Synuclein (Ser129) (D1R1R) Rabbit mAb (upper), or α-Synuclein (D37A6) XP® Rabbit mAb #4179 (lower) (green), in the presence of Phospho-α-Synuclein (Ser129) peptide (middle) or Non-phospho-α-Synuclein (Ser129) peptide (right). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Learn more about how we get our imagesConfocal immunofluorescent analysis of untreated (left) or λ phosphatase-treated (right) mouse hippocampus using Phospho-α-Synuclein (Ser129) (D1R1R) Rabbit mAb (upper), or α-Synuclein (D37A6) XP® Rabbit mAb #4179 (lower) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Learn more about how we get our imagesConfocal immunofluorescent analysis of mouse hippocampus using Phospho-α-Synuclein (Ser129) (D1R1R) Rabbit mAb (green). High-magnification showing punctate and sparse nuclear labeling (arrows). Specificity of nuclear staining is unclear (see Specificity/Sensitivity statement and supporting IF-F validation data). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Learn more about how we get our imagesFor 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 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 October 2017
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) | 1:1000 |
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.
Phospho-α-Synuclein (Ser129) (D1R1R) Rabbit mAb recognizes endogenous levels of α-Synuclein protein only when phosphorylated at Ser129. Specificity of nuclear staining is unclear but consistent with previous publications (4).
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser129 of human α-Synuclein protein.
α-Synuclein is a protein of 140-amino acids expressed abundantly in the brain. α-Synuclein is also the main component of pathogenic Lewy bodies and Lewy neurites. Research studies have shown that mutations of the α-synuclein gene are linked to Parkinson's disease (1).
Various research studies have shown that phosphorylation of α-Synuclein at Ser129 is a highly toxic event causing degeneration of dopaminergic neurons associated with Parkinson's disease. This is proposed to occur through increased misfolding, aggregation, and accumulation of α-Synuclein phosphorylated at this site (2). GSK-3β is one of several kinases that has been reported to phosphorylate α-Synuclein at Ser129 (3).
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. Tween is a registered trademark of ICI Americas, Inc.
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23706S | 100 µl (10 western blots) | $ 297.0 |