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Purified Mouse Anti-Phospholipase Cγ1
Purified Mouse Anti-Phospholipase Cγ1

Western blot analysis of Phospholipase Cγ1 on a A431 cell lysate (Human epithelial carcinoma; ATCC CRL-1555). Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the mouse anti-Phospholipase Cγ1 antibody.

Purified Mouse Anti-Phospholipase Cγ1

Immunofluorescence staining of HeLa cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10,000 cells per well.  After overnight incubation, cells were stained using the methanol fix/perm protocol (see Recommended Assay Procedure) and the purified mouse anti-Phospholipase Cγ1 antibody.  The second step reagent was FITC goat anti-mouse Ig (Cat. No. 554001).  The image was taken on a BD Pathway™ 850 imager using a 20x objective.  This antibody also stained A549 and U2OS cells and can be used with either fix/perm protocol (see Recommended Assay Procedure).

Western blot analysis of Phospholipase Cγ1 on a A431 cell lysate (Human epithelial carcinoma; ATCC CRL-1555). Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the mouse anti-Phospholipase Cγ1 antibody.

Immunofluorescence staining of HeLa cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10,000 cells per well.  After overnight incubation, cells were stained using the methanol fix/perm protocol (see Recommended Assay Procedure) and the purified mouse anti-Phospholipase Cγ1 antibody.  The second step reagent was FITC goat anti-mouse Ig (Cat. No. 554001).  The image was taken on a BD Pathway™ 850 imager using a 20x objective.  This antibody also stained A549 and U2OS cells and can be used with either fix/perm protocol (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing), Mouse, Rat, Dog, Chicken (Tested in Development)
Mouse IgG1
Cow PLCγ1 N-terminal region Peptide
Western blot (Routinely Tested), Bioimaging, Immunohistochemistry, Immunoprecipitation (Tested During Development)
148 kDa
250 µg/ml
AB_397446
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

Western blot:  Please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

Recommended Protocol for Bioimaging:

1.        Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and

        culture overnight to 48 hours.

2.        Remove the culture medium from the wells, and wash (one to two times) with 100 µl of 1× PBS.

3.        Fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and

        incubating for 10 minutes at room temperature (RT).

4.        Remove the fixative from the wells, and wash the wells (one to two times) with 100 µl of 1× PBS.

5.        Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c):

                a.        Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubate for 5

                        minutes at RT.

                b.        Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

                c.        Add 100 µl of 1× Perm/Wash buffer (Cat. No. 554723) to each well and incubate for 15 to 30 minutes at RT.  Continue to use 1×

                        Perm/Wash buffer for all subsequent wash and dilutions steps.

6.        Remove the permeabilization buffer from the wells, and wash one to two times with 100 µl of appropriate buffer (either 1× PBS or 1×

        Perm/Wash buffer, see step 5.c.).

7.        Optional blocking step:  Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer BD Pharmingen™ Stain Buffer

        (FBS) (Cat. No. 554656) or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.

8.        Dilute the antibody to its optimal working concentration in appropriate dilution buffer.  Titrate purified (unconjugated) antibodies and

        second-step reagents to determine the optimal concentration.  If using a Bioimaging Certified antibody conjugate, dilute it 1:10.

9.        Add 50 µl of diluted antibody per well and incubate for 120 minutes at RT.  Incubate in the dark if using fluorescently labeled antibodies.

10.        Remove the antibody, and wash the wells three times with 100 µl of wash buffer.  An optional detergent wash (100 µl of 0.05% Tween in 1×

        PBS) can be included prior to the regular wash steps.

11.   If the antibody being used is fluorescently labeled, then move to step 12.  Otherwise, if using a purified unlabeled antibody, repeat steps 8 to

        10 with a fluorescently labeled second-step reagent to detect the purified antibody.

12.        After the final wash, counter-stain the nuclei by adding 100 µl of a 2 µg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261)

        in 1× PBS to each well at least 15 minutes before imaging.

13.        View and analyze the cells on an appropriate imaging instrument. Recommended filters for the BD Pathway™ instruments are:

Instrument                        Excitation        Emission        Dichroic

BD Pathway 855                488/10                515 LP                Fura/FITC

BD Pathway 435                482/35                536/40                FF506

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
610027 Rev. 2
Antibody Details
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10/PLCgamma

The Phospholipase C (PLC) isozymes hydrolyze phosphatidyl inositol biphosphate to inositol triphosphate and diacylglycerol.  The former causes release of calcium from the endoplasmic reticulum, while the latter is an activator of Protein Kinase C.  Within the PLC family, PLCγ is the only member that contains SH2 and SH3 domains.  These domains enable it to interact with receptor tyrosine kinases and become enzymatically activated via phosphorylation.  It exists as two isoforms: 1) PLCγ1, which is ubiquitously expressed, and 2) PLCγ2, found primarily in the lymphoid system.  PLCγ is essential for growth factor-induced cell motility and mitogenesis.  PLCγ1 null mice exhibit retarded embryonic growth and lethality in midgestation.  Overexpression of PLCγ is evident in several forms of cancer, and it has been identified as a key mediator of PDGF-dependent cellular transformation.  Thus regulation of PLCγ activity by growth factors is involved in cell growth and transformation.

The 10/PLCgamma monoclonal antibody recognizes PLCγ1, regardless of phosphorylation status.  It does not cross-react with PLCγ2.

610027 Rev. 2
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
610027 Rev.2
Citations & References
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Development References (3)

  1. Dayanir V, Meyer RD, Lashkari K, Rahimi N. Identification of tyrosine residues in vascular endothelial growth factor receptor-2/FLK-1 involved in activation of phosphatidylinositol 3-kinase and cell proliferation. J Biol Chem. 2001; 276(21):17686-17692. (Biology: Immunoprecipitation, Western blot). View Reference
  2. Obermeier A, Tinhofer I, Grunicke HH, Ullrich A. Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation. EMBO J. 1996; 15(1):73-82. (Biology). View Reference
  3. Vossmeyer D, Hofmann W, Loster K, Reutter W, Danker K. Phospholipase Cgamma binds alpha1beta1 integrin and modulates alpha1beta1 integrin-specific adhesion. J Biol Chem. 2002; 277(7):4636-4643. (Biology: Immunoprecipitation, Western blot). View Reference
610027 Rev. 2

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


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Non-IVD products are For Research Use Only. Not for use in diagnostic or therapeutic procedures.

 

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