BP7149 Phospholipase D1 (PLD1) (N-term) antibody

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Rabbit anti Human Phospholipase D1 (PLD1)


Product Description for Phospholipase D1 (PLD1)

Rabbit anti Human Phospholipase D1 (PLD1).
Properties: (N-term)
Presentation: Aff - Purified
Product is tested for Western blot / Immunoblot.

Properties for Phospholipase D1 (PLD1)

Product Category Primary Antibodies
Quantity 0.1 mg
Synonyms PL-D1
Presentation Aff - Purified
Reactivity Hu
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

A chemically synthesized peptide derived from the N-terminal region of human PC-specific PL-D1 protein
Property N-term
Application Western blotting: use at a dilution of 1:100 to 1:2000 against purified recombinant protein. Due to the low expression levels of this protein, it is suggested that the protein be immunoprecipitated before Western blotting.
Background 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. PL-D1 is regulated by GTP-binding proteins, (ARF and Rho families) and by protein kinase C.
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 μg 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.
7. Soak the PVDF 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) overnight at 4°C.
12. Incubate the blocked blot with primary antibody at appropriate concentration in Tris buffered saline supplemented with 3% BSA and 0.1% Tween 20 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 Na3VO4
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 μg/mL aprotinin
10 μg/mL leupeptin
1 μg/mL pepstatin
(alternatively, protease inhibitor cocktail such as Sigma catalog number 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 Hammond S.M., et al. (1997) Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha. J. Biol. Chem. 272 (6):3860-3868.
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 (cites the use of the PL-D1 and PL-D2 antibodies).
Zhong, M., et al. (2002) Elevated phospholipase D activity induced apoptosis in normal rat fibroblasts. Biochem. Phys. Res. Comm. 298:474-477 (cites the use of the PL-D1 and PL-D2 antibodies).
Storage Store (in aliquots ) at -20°C. Avoid repeated freezing and thawing.
Shelf life: one year from despatch.
Peptide affinity chromatography
Buffer System:
PBS, pH 7.2, with 0.1% BSA and 0.09% sodium azide
Aff - Purified
This antibody specifically recognizes the N-terminal region of the PL-D1 enzyme.

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