BP7151 Phospholipase D2 (PLD2) (N-term) antibody

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Rabbit anti Mouse Phospholipase D2 (PLD2)


Product Description for Phospholipase D2 (PLD2)

Rabbit anti Mouse Phospholipase D2 (PLD2).
Properties: (N-term)
Presentation: Aff - Purified
Product is tested for Western blot / Immunoblot.

Properties for Phospholipase D2 (PLD2)

Product Category Primary Antibodies
Quantity 0.1 mg
Synonyms PL-D2
Presentation Aff - Purified
Reactivity Ms
Applications WB
Clonality Polyclonal
Host Rabbit
Shipping to Worldwide
PDF datasheet View Datasheet
Manufacturer Acris Antibodies GmbH
Material safety datasheet MSDS for Polyclonal Antibodies (de)

Datasheet Extract

Synthetic peptide derived from the N-terminal region of mouse PC-specific PL-D2 protein
Property N-term
Application Western blotting: We recommend using this product at a dilution of 1:100 to 1:2000 against recombinant purified protein. Due to the low expression levels of this protein, it is suggested that the protein be immunoprecipitated before Western blotting.
Concentration 0.5 mg/ml
Protocols Western Blotting Procedure

1. Lyse approximately 10e7 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application.
2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifuged at 100,000 x g for 30 minutes for greater clarification.
3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Polypropylene tubes are recommended for storing cell lysates.
4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100°C.
5. Load 10-30 ug of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by SDS-PAGE.
6. In preparation for the Western transfer, cut a piece of PVDF membrane slightly larger than the gel. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. Alternatively, nitrocellulose may be used.
7. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes.
8. Assemble the gel and membrane into the sandwich apparatus.
9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature.
10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes.
11. Block the membrane with blocking buffer (formulation provided below) for one hour at room temperature or overnight at 4°C.
12. Incubate the blocked blot with primary antibody at a 1:1000 dilution in Tris buffered saline supplemented with 3% BSA and 0.1% Tween 20 overnight at 4oC or for two hours at room temperature.
13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20.
14. Detect the antibody band using an appropriate secondary antibody, such as goat F(ab')2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab')2 anti-rabbit IgG horseradish peroxidase conjugate in conjunction with your chemiluminescence reagents and instrumentation.

Cell Lysis Buffer Formulation:
10 mM Tris, pH 7.4
100 mM NaCl
1 mM NaF
20 mM Na4P2O7
2 mM NaVO4
0.1% SDS
0.5% sodium deoxycholate
1% Triton-X 100
10% glycerol
1 mM PMSF (made from a 0.3 M stock in DMSO) or 1 mM AEBSF (water soluble version of PMSF)
60 ug/mL aprotinin
10 ug/mL leupeptin
1 ug/mL pepstatin
(alternatively, protease inhibitor cocktail such as Sigma Cat. # P2714 may be used)

Transfer Buffer Formulation:
2.4 gm Tris base
14.2 gm glycine
200 mL methanol
Q.S. to 1 liter, then add 1 mL 10% SDS.
Cool to 4°C prior to use.

Tris Buffered Saline Formulation:
20 mM Tris-HCl, pH 7.4
0.9% NaCl

Blocking Buffer Formulation:
100 mL Tris buffered saline
5 gm BSA
0.1 mL Tween 20
General Readings Colley, et al. (1997) Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization. Curr. Biol. 7:191-201
Sciorra, V.A. and A.J. Morris (1999) Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells. Mol. Biol. Cell 10:3863-3876
Zhong, M., et al. (2002) Elevated phospholipase D activity induced apoptosis in normal rat fibroblasts. Biochem. Phys. Res. Comm. 298:474-477
Storage Store at 2-8°C short term only. Aliquot and store at -20°C to -80°C for longer. Avoid repeated freezing and thawing. Shelf life: One year from despatch.
Peptide affinity chromatography
Buffer System:
Dulbecco's phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), with 1.0 mg/mL BSA (IgG, protease free) as a carrier and 0.05% sodium azide as preservative
Aff - Purified
This antibody specifically recognizes the N-terminal region of the PL-D2 protein. Activation of PL-D results in the generation of second messengers, phosphatidic acid and diglycerides, and appears to be involved in secretion, vesicle trafficking, mitosis and meiosis. In leukocytes, PL-D regulates cytoskeletal-dependent antimicrobial responses such as phagocytosis and oxidant generation. The mechanisms that regulate PL-D2 are not known. This isoform is unaffected by the activators of PL-D1.

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