REACTIVITY | SENSITIVITY | MW (kDa) | SOURCE |
---|---|---|---|
H M R Mk Dm | Endogenous | 36, 38 | Rabbit |
Western blot analysis of extracts from HeLa, NIH/3T3, C6 and COS cells using PP2A C Subunit Antibody.
Learn more about how we get our imagesImmunohistochemical analysis of paraffin-embedded human breast carcinoma using PP2A C Subunit Antibody in the presence of control peptide (left) or PP2A C Subunit Blocking Peptide, #1067 (right).
Learn more about how we get our imagesImmunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization, using PP2A C Subunit Antibody.
Learn more about how we get our imagesImmunohistochemical analysis of paraffin-embedded human colon carcinoma using PP2A C Subunit Antibody.
Learn more about how we get our imagesImmunohistochemical analysis of paraffin-embedded human lung carcinoma using PP2A C Subunit Antibody in the presence of control peptide (left) or antigen-specific peptide, #1067 (right).
Learn more about how we get our imagesImmunofluorescent analysis of paraformaldehyde fixed HeLa cells using PP2A C Subunit Antibody.
Learn more about how we get our imagesFlow cytometric analysis of NIH/3T3 cells using PP2A C Subunit Antibody (blue) compared to a non-specifc negative control antibody (red).
Learn more about how we get our imagesFor western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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, 10 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 June 2016
Protocol Id: 263
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: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted June 2005
revised March 2016
Protocol Id: 303
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a 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 November 2013
Protocol Id: 24
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2017
Protocol Id: 404
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
Immunohistochemistry (Paraffin) | 1:100 |
Immunofluorescence (Immunocytochemistry) | 1:50 |
Flow Cytometry | 1:100 |
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.
This antibody detects endogenous levels of PP2A catalytic subunit protein (both α and β isoforms). The antibody does not cross-react with other PP2A subunits.
Human, Mouse, Rat, Monkey, D. melanogaster
Chicken, Pig
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino acids at the carboxy terminus of human PP2A catalytic subunit. Antibodies are purified by protein A and peptide affinity chromatography.
Protein phosphatase type 2A (PP2A) is an essential protein serine/threonine phosphatase that is conserved in all eukaryotes. PP2A is a key enzyme within various signal transduction pathways as it regulates fundamental cellular activities such as DNA replication, transcription, translation, metabolism, cell cycle progression, cell division, apoptosis and development (1-3). The core enzyme consists of catalytic C and regulatory A (or PR65) subunits, with each subunit represented by α and β isoforms (1). Additional regulatory subunits belong to four different families of unrelated proteins. Both the B (or PR55) and B' regulatory protein families contain α, β, γ and δ isoforms, with the B' family also including an ε protein. B'' family proteins include PR72, PR130, PR59 and PR48 isoforms, while striatin (PR110) and SG2NA (PR93) are both members of the B''' regulatory protein family. These B subunits competitively bind to a shared binding site on the core A subunit (1). This variable array of holoenzyme components, particularly regulatory B subunits, allows PP2A to act in a diverse set of functions. PP2A function is regulated by expression, localization, holoenzyme composition and post-translational modification. Phosphorylation of PP2A at Tyr307 by Src occurs in response to EGF or insulin and results in a substantial reduction of PP2A activity (4). Reversible methylation on the carboxyl group of Leu309 of PP2A has been observed (5,6). Methylation alters the conformation of PP2A, as well as its localization and association with B regulatory subunits (6-8).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Explore pathways related to this product.
Product # | Size | Price |
---|---|---|
2038T | 20 µl (2 western blots) | $ 109.0 |
2038S | 100 µl (10 western blots) | $ 255.0 |