REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H M Mk | Endogenous | 150 | Rabbit IgG |
Western blot analysis of extracts from serum-starved A-431 and A549 cells, untreated (-) or treated (+) with hEGF #8916 (100 ng/mL, 15 min) or serum-starved NIH/3T3 cells, untreated (-) or treated (+) with hPDGF-BB #8912 (50 ng/mL, 15 min), using Phospho-PLCγ1 (Ser1248) (D25A9) Rabbit mAb (upper) or PLCγ1 (D9H10) XP® Rabbit mAb #5690 (lower).
Learn more about how we get our images.Immunoprecipitation (IP)/Western blot analysis of extracts from serum-starved HeLa cells, untreated (-) or treated (+) with TPA #4174 (100 nM, 15 min) prior to lysis in SDS (lanes 1 and 2) or IP lysis buffer (lane 3, TPA-treated only). IP Lysates were then subjected to immunoprecipitation with Phospho-PLCγ1 (Ser1248) (D25A9) Rabbit mAb (lane 4), PLCγ1 (D9H10) XP® Rabbit mAb #5690 (lane 5), or Normal Rabbit IgG #2729 (lane 6). The western blot was probed using Phospho-PLCγ1 (Ser1248) (D25A9) Rabbit mAb. Lane 3 represents 10% input.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human colon (normal adjacent to tumor) using Phospho-PLCγ1 (Ser1248) (D25A9) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-PLCγ1 (Ser1248) (D25A9) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of SignalSlide® Phospho-EGF Receptor IHC Controls #8102 [paraffin-embedded KYSE450 cell pellets untreated (left) or EGF-treated (right)] using Phospho-PLCγ1 (Ser1248) (D25A9) Rabbit mAb.
Learn more about how we get our images.Confocal immunofluorescent analysis of A-431 cells, serum starved (left) or treated with hEGF #8916 (100 ng/mL for 15 min) using Phospho-PLCγ1 (Ser1248) (D25A9) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Learn more about how we get our images.Flow cytometric analysis of Jurkat cells, treated with U0126 #9903 (blue) or TPA #4174 (green), using Phospho-PLCγ1 (Ser1248) (D25A9) Rabbit mAb.
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.
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 February 2010
revised March 2016
Protocol Id: 300
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 | 1:50 |
Immunohistochemistry (Paraffin) | 1:200 |
Immunofluorescence (Immunocytochemistry) | 1:100 |
Flow Cytometry | 1:200 |
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-PLCγ1 (Ser1248) (D25A9) Rabbit mAb recognizes endogenous levels of PLCγ1 protein only when phosphorylated at Ser1248.
Human, Mouse, Monkey
Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser1248 of human PLCγ1 protein.
Phosphoinositide-specific phospholipase C (PLC) plays a significant role in transmembrane signaling. In response to extracellular stimuli such as hormones, growth factors, and neurotransmitters, PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two secondary messengers: inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (1). At least four families of PLCs have been identified: PLCβ, PLCγ, PLCδ, and PLCε. Phosphorylation is one of the key mechanisms that regulate the activity of PLC. PLCγ is activated by both receptor and non-receptor tyrosine kinases (2). PLCγ forms a complex with EGF and PDGF receptors, which leads to the phosphorylation of PLCγ at Tyr771, 783, and 1248 (3). Phosphorylation by Syk at Tyr783 activates the enzymatic activity of PLCγ1 (4). PLCγ2 is engaged in antigen-dependent signaling in B cells and collagen-dependent signaling in platelets. Phosphorylation by Btk or Lck at Tyr753, 759, 1197, and 1217 is correlated with PLCγ2 activity (5,6).
Two mammalian PLCγ isoforms (γ1 and γ2) have been cloned and characterized (7,8). Like other PLC-family members, PLCγ1 and PLCγ2 contain calcium-binding (EF-hand, C2) and lipid-interacting (PH, EF-hand) domains necessary for their enzymatic activity and substrate recognition. Uniquely, PLCγ isoforms have additional, conserved SH2 and SH3 domains critical for their functions as signaling molecules and scaffolding proteins. Upon growth factor stimulation, PLCγ1 is recruited (via SH2 domains) to phosphotyrosine residues within the cytoplasmic tail of many RTKs where it serves as a substrate for the RTK and provides docking sites for additional proteins involved in RTK signaling (4-6,9-12). PLCγ1 and γ2 can also be activated downstream of receptors lacking intrinsic tyrosine kinase activity. This has been reported downstream of multiple G protein-coupled receptors and the T cell receptor in which tyrosine kinases of the Src, Syk, and Tec families serve to bind, phosphorylate, and activate PLCγ (reviewed in 13-15). Phosphorylation at tyrosine residues by both receptor and non-receptor tyrosine kinases results in robust activation of PLCγ1 activity, leading to generation of second messengers. In response to agonists, PLCγ1 is phosphorylated on Tyr783, Tyr711, and Tyr1253 (Tyr753, Tyr759, and Tyr1217 in PLCγ2) resulting in robust PI-4,5-P2 hydrolysis (4-6,9-12). Interestingly recent evidence suggests a role for tyrosine kinase-independent regulation of PLCγ in some systems. For example, in response to EGF, proline-rich regions of Akt interact with the SH3 domain of PLCγ1 resulting in association of the two enzymes, phosphorylation of PLCγ1 at Ser1248, and enhanced cellular motility (16). This finding demonstrates that PLCγ1 can function as a "scaffold" between RTKs and Akt, thereby establishing a mechanism by which the Akt signaling pathway cross-talks with tyrosine kinases. However, the mechanism and functional significance of phosphorylation at Ser1248 remains to be fully clarified, as it has also been shown that PKA-mediated phosphorylation at this site is inhibitory to PLCγ1 tyrosine phosphorylation and phospholipase activity in CD3-treated Jurkat cells (17), suggesting that Ser1248 may be an allosteric regulator of PLCγ1 activity.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. DRAQ5 is a registered trademark of Biostatus Limited.
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