Revision 1
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
Caspase-3 (8G10) Rabbit mAb 9665 40 µl 17, 19, 35 kDa Rabbit IgG
Caspase-6 Antibody 9762 40 µl 15, 35 kDa Rabbit 
Caspase-7 (D2Q3L) Rabbit mAb 12827 40 µl 20, 35 kDa Rabbit IgG
Lamin A/C (4C11) Mouse mAb 4777 40 µl 74 (Lamin A), 63 (Lamin C) kDa Mouse IgG2a
Lamin B1 (D4Q4Z) Rabbit mAb 12586 40 µl 68, 25 kDa Rabbit IgG
PARP Antibody 9542 40 µl 89, 116 kDa Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl Horse 

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

Description

The Effector Caspases and Substrates Antibody Sampler Kit provides an economical means to evaluate the activation of effector (executioner) caspases. The kit contains enough primary antibody to perform at least four western blots per 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

Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Caspase-3 (CPP-32, Apoptain, Yama, SCA-1), Caspase-6 (Mch2), and Caspase-7 (CMH-1, Mch3, ICE-LAP3) are effector caspases functioning in cellular apoptotic processes (1-6). Upon apoptotic stimulation, initiator caspases such as caspase-9 (ICE-LAP6, Mch6) are cleaved and activated (7). The activated upstream caspases further process downstream executioner caspases by cleaving them into activated large and small subunits, thereby initiating a caspase cascade leading to apoptosis (4,6,8-10).

PARP, a 116 kDa nuclear poly (ADP-ribose) polymerase, appears to be involved in DNA repair in response to environmental stress (11). This protein can be cleaved by many ICE-like caspases in vitro (1,12) and is one of the main cleavage targets of caspase-3 in vivo (10,13). In human PARP, cleavage occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (10,12). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (14).

Lamins are nuclear membrane structural components that are important in maintaining normal cell functions, such as cell cycle control, DNA replication, and chromatin organization (15-17). Lamins have been subdivided into types A and B. Type-A lamins consist of lamin A and C, which arise from alternative splicing of the lamin A gene LMNA. Lamin A and C are cleaved by caspases into large (41-50 kDa) and small (28 kDa) fragments, which can be used as markers for apoptosis (18,19). Type-B lamins consist of lamin B1 and B2, encoded by separate genes (20-22). Lamin B1 is also cleaved by caspases during apoptosis (23).

  1. Cohen, G.M. (1997) Biochem J 326 ( Pt 1), 1-16.
  2. Fernandes-Alnemri, T. et al. (1994) J Biol Chem 269, 30761-4.
  3. Faleiro, L. et al. (1997) EMBO J 16, 2271-81.
  4. Fernandes-Alnemri, T. et al. (1995) Cancer Res 55, 6045-52.
  5. Duan, H. et al. (1996) J Biol Chem 271, 1621-5.
  6. Lippke, J.A. et al. (1996) J Biol Chem 271, 1825-8.
  7. Li, P. et al. (1997) Cell 91, 479-89.
  8. Slee, E.A. et al. (1999) J Cell Biol 144, 281-92.
  9. MacFarlane, M. et al. (1997) J Cell Biol 137, 469-79.
  10. Nicholson, D.W. et al. (1995) Nature 376, 37-43.
  11. Satoh, M.S. and Lindahl, T. (1992) Nature 356, 356-8.
  12. Lazebnik, Y.A. et al. (1994) Nature 371, 346-7.
  13. Tewari, M. et al. (1995) Cell 81, 801-9.
  14. Oliver, F.J. et al. (1998) J Biol Chem 273, 33533-9.
  15. Dunbar, J.C. and Lu, H. (2000) Brain Res Bull 52, 123-6.
  16. Goldberg, M. et al. (1999) Crit Rev Eukaryot Gene Expr 9, 285-93.
  17. Yabuki, M. et al. (1999) Physiol Chem Phys Med NMR 31, 77-84.
  18. Rao, L. et al. (1996) J Cell Biol 135, 1441-55.
  19. Orth, K. et al. (1996) J Biol Chem 271, 16443-6.
  20. Biamonti, G. et al. (1992) Mol Cell Biol 12, 3499-506.
  21. Lin, F. and Worman, H.J. (1995) Genomics 27, 230-6.
  22. Pollard, K.M. et al. (1990) Mol Cell Biol 10, 2164-75.
  23. Chandler, J.M. et al. (1997) Biochem J 322 ( Pt 1), 19-23.

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.

    使用に関する制限

    法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

    研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #12690

    Effector Caspases and Substrates Antibody Sampler Kit

    Effector Caspases and Substrates Antibody Sampler Kit: Image 1 Expand Image
    Flow cytometric analysis of Daudi cells (blue) and MJ cells (green) using Lamin A/C (4C11) Mouse mAb (solid lines) or concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from serum-starved HeLa cells treated with Staurosporine (1 uM, 3 hours) using PARP Antibody #9542. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 3 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from Jurkat cells treated with Cytochrome C using Caspase-6 Antibody #9762. The virtual lane view (left) shows the target bands (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from HeLa cells (lane 1) or LMNB1 knock-out cells (lane 2) using Lamin B1 (D4Q4Z) Rabbit mAb #12586 (upper), and β-actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the LMNB1 knock-out HeLa cells confirms specificity of the antibody for LMNB1.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from Jurkat and A20 cells, untreated (-) or treated with Etoposide #2200 (25 μM, overnight; +), using Caspase-7 (D2Q3L) Rabbit mAb.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or HeLa cells with an apparent in-frame truncation mutation in the gene encoding LMNA (lane 2) using Lamin A/C (4C11) Mouse mAb #4777 (upper) or α-actinin (D6F6) XP® Rabbit mAb #6487 (lower). The change in LMNA molecular weight in the mutated HeLa cells is consistent with an in-frame deletion.
    Effector Caspases and Substrates 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.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 8 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.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from NIH/3T3 cells, untreated or staurosporine-treated (1 µM), and Jurkat cells, untreated or etoposide-treated (25 µM), using PARP Antibody.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® Caspase-3 siRNA II (+), using Caspase-3 (8G10) Rabbit mAb and α-Tubulin (11H10) Rabbit mAb #2125. Caspase-3 (8G10) Rabbit mAb confirms silencing of caspase-3 expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of caspase-3 siRNA.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell lines, untreated or cytochrome c treated (1hr, 0.25 mg/ml in vitro) using Caspase-6 Antibody #9762 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Effector Caspases and Substrates Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from various cell lines using Lamin B1 (D4Q4Z) Rabbit mAb.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from various cell lines using Lamin A/C (4C11) Mouse mAb.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® Caspase-3 siRNA I (+), using Caspase-3 (8G10) Rabbit mAb and p42 MAPK Antibody #9108. Caspase-3 (8G10) Rabbit mAb confirms silencing of caspase-3 expression, while the p42 MAPK Antibody is used to control for loading and specificity of caspase-3 siRNA.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from HeLa cells, untreated or treated with Staurosporine #9953 (1 μM, 3 hr), using Lamin B1 (D4Q4Z) Rabbit mAb.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from THP-1 cells, untreated or treated with cycloheximide (CHX, 10 μg/ml, overnight) followed by TNF-α #8902 (20 ng/ml, 4 hr), using Lamin A/C (4C11) Mouse mAb.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of HeLa (human) and NIH/3T3 (mouse) cell extracts, untreated and treated with 1 μM staurosporine (3 hr) in vivo, using Caspase-3 (8G10) Rabbit mAb.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Lamin A/C (4C11) Mouse mAb.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 19 Expand Image
    Immunoprecipitation of cleaved caspase-3 from Jurkat cell extracts untreated (control) or treated with etoposide (25uM 5hrs) (apoptotic) using Caspase-3 (8G10) Rabbit mAb, and western probed with the same antibody.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Lamin A/C (4C11) Mouse mAb.
    Effector Caspases and Substrates Antibody Sampler Kit: Image 21 Expand Image
    Immunofluorescent analysis of normal rat brain using Lamin A/C (4C11) Mouse mAb (green) and MAP2 Antibody #4542 (red).
    Effector Caspases and Substrates Antibody Sampler Kit: Image 22 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Lamin A/C (4C11) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
    Effector Caspases and Substrates Antibody Sampler Kit: Image 23 Expand Image
    Flow cytometric analysis of HeLa cells (green) using Lamin A/C (4C11) Mouse mAb (solid lines) or a concentration matched Mouse (G3A1) mAb IgG Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
    Image Gallery
    Learn more about how we get our images
    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.