Revision 4

#8334Store at -20C

1 Kit

(5 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-HSL (Ser563) Antibody 4139 20 µl 81, 83 kDa Rabbit 
Phospho-HSL (Ser565) Antibody 4137 20 µl 81, 83 kDa Rabbit 
Perilipin-1 (D1D8) XP® Rabbit mAb 9349 20 µl 62 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
HSL (D6W5S) XP® Rabbit mAb 18381 20 µl 81, 83 kDa Rabbit IgG
Phospho-HSL (Ser660) Antibody 45804 20 µl 81, 83 kDa Rabbit 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Lipolysis Activation Antibody Sampler Kit provides an economical means to evaluate the activation status of multiple members of the lipolysis pathway, including phosphorylated HSL and perilipin. The kit includes enough antibody to perform two western mini-blot experiments with each primary antibody.

Storage

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.

Background

Triacylglycerol is stored in lipid droplets as a primary energy reserve. During lipolysis, triacylglycerols in adipocytes are hydrolyzed into free fatty acids and glycerol. Perilipin, localized at the periphery of lipid droplets, serves as a protective coating against lipases (1-3). Evidence suggests that PKA regulates lipolysis by phosphorylating perilipin and hormone-sensitive lipase (HSL) (1,2,4,5). Phosphorylation of perilipin results in the conformational change that exposes lipid droplets to endogenous lipases, such as HSL (2). Phosphorylation of HSL at Ser563, Ser659, and Ser660 by PKA stimulates HSL activity, which in turn catalyzes the hydrolysis of triacylglycerol (6,7).

  1. Greenberg, A.S. et al. (1991) J Biol Chem 266, 11341-6.
  2. Brasaemle, D.L. (2007) J Lipid Res 48, 2547-59.
  3. Ducharme, N.A. and Bickel, P.E. (2008) Endocrinology 149, 942-9.
  4. Egan, J.J. et al. (1990) J Biol Chem 265, 18769-75.
  5. Brasaemle, D.L. et al. (2009) Mol Cell Biochem 326, 15-21.
  6. Degerman, E. et al. (1990) Proc Natl Acad Sci U S A 87, 533-7.
  7. Anthonsen, M.W. et al. (1998) J Biol Chem 273, 215-21.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 4
    #8334

    Lipolysis Activation Antibody Sampler Kit

    Lipolysis Activation Antibody Sampler Kit: Image 1 Expand Image
    Confocal immunofluorescent analysis of 10-day differentiated 3T3-L1 cells (left, positive) or undifferentiated 3T3-L1 cells (right, negative) using HSL (D6W5S) XP® Rabbit mAb (green), Perilipin-1 (D1D8) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #52975 (red), and DAPI #4083 (blue).
    Lipolysis Activation Antibody Sampler Kit: Image 2 Expand Image
    Immunoprecipitation of perilipin-1 protein from 3T3-L1 cell extracts differentiated for 4 days. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Perilipin-1 (D1D8) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as the secondary antibodies.
    Lipolysis Activation Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from undifferentiated and differentiated 3T3-L1 cells using HSL (D6W5S) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Lipolysis Activation Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from differentiated 3T3-L1 cells treated with forskolin, oligomycin or lambda protein phosphatase, using Phospho-HSL (Ser565) Antibody.
    Lipolysis Activation Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from differentiated 3T3-L1 cells treated with isoproterenol or lambda protein phosphatase, using Phospho-HSL (Ser563) Antibody.
    Lipolysis Activation Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from 3T3-L1 cells, undifferentiated (lane 1) or differentiated 4 days into adipocytes and treated with isoproterenol (10 μM, 20 min; lane 2), using Phospho-HSL (Ser660) Antibody (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
    Lipolysis Activation Antibody Sampler Kit: Image 7 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Lipolysis Activation Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from human pre-adipocytes and adipocytes using Perilipin-1 (D1D8) XP® Rabbit mAb (upper) and β-Actin Antibody #4967 (lower).
    Lipolysis Activation Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from human pre-adipocytes and differentiated human adipocytes using HSL (D6W5S) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Lipolysis Activation Antibody Sampler Kit: Image 10 Expand Image
    Confocal immunofluorescent analysis of 3T3-L1 cells, untreated (left) or phosphatase-treated (right), labeled with Phospho-HSL (Ser565) Antibody (red) showing cytoplasmic localization in differentiated cells. Lipid droplets have been labeled with BODIPY® 493/503 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Lipolysis Activation Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from 3T3-L1 cells, undifferentiated (lane 1) or differentiated 4 days into adipocytes and treated with isoproterenol (10 μM, 20 min; lane 2), using Phospho-HSL (Ser660) Antibody. Phospho-specificity was verified by incubating the antibody without peptide (left), with HSL (Ser660) non-phosphopeptide (middle), or with HSL (Ser660) phosphopeptide (right) prior to incubation with the membrane.
    Lipolysis Activation Antibody Sampler Kit: Image 12 Expand Image
    Immunohistochemical analysis of paraffin-embedded breast carcinoma using Perilipin-1 (D1D8) XP® Rabbit mAb. Note specific staining of adipocytes.
    Lipolysis Activation Antibody Sampler Kit: Image 13 Expand Image
    Immunoprecipitation of HSL from differentiated human adipocyte extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is HSL (D6W5S) XP® Rabbit mAb. Western blot analysis was performed usingHSL (D6W5S) XP® Rabbit mAb.
    Lipolysis Activation Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of parafin-embedded mouse brown fat using Perilipin-1 (D1D8) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Lipolysis Activation Antibody Sampler Kit: Image 15 Expand Image
    Confocal immunofluorescent analysis of differentiated human adipocytes (left) or human pre-adipocytes (right) using HSL (D6W5S) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight® 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Lipolysis Activation Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of parafin-embedded mouse kidney with staining of adjacent adipose tissue using Perilipin-1 (D1D8) XP® Rabbit mAb.
    Lipolysis Activation Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of parafin-embedded mouse prostate with staining of adjacent adipose tissue using Perilipin-1 (D1D8) XP® Rabbit mAb.
    Lipolysis Activation Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of parafin-embedded mouse testis with staining of adjacent adipose tissue using Perilipin-1 (D1D8) XP® Rabbit mAb.
    Lipolysis Activation Antibody Sampler Kit: Image 19 Expand Image
    Confocal immunofluorescent analysis of frozen mouse brown adipose tissue using Perilipin-1 (D1D8) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Lipolysis Activation Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of 8-day differentiated 3T3-L1 cells, using Perilipin (D1D8) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).