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17229
Pro-Survival Bcl-2 Family Antibody Sampler Kit II
Primary Antibodies
Antibody Sampler Kit

Pro-Survival Bcl-2 Family Antibody Sampler Kit II #17229

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Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 1

Western blot analysis of control HeLa cells (lane 1) or Bcl-2 knockout HeLa cells (lane 2) using Bcl-2 (D55G8) Rabbit mAb #4223 (upper), or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the Bcl-2 knockout HeLa cells confirms the specificity of the antibody for Bcl-2.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 2

Flow cytometric analysis of Jurkat cells using Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb and Propidium Iodide (PI)/RNase Staining Solution #4087 to measure DNA content. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 3

Flow cytometric analysis of SK-OV-3 cells (blue) and MCF7 cells (green) using Mcl-1 (D2W9E) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 4

Immunoprecipitation of phospho-Mcl-1 (Thr163) from KARPAS-299 cells treated with TPA #4174 (200 nM, 30 min) using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Mcl-1 (Thr163) (D5M9D) Rabbit mAb (lane 3). Lane 1 represents 10% input. Western blot was perform using Phospho-Mcl-1 (Thr163) (D5M9D) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody to avoid cross reactivity with IgG heavy and light chains. KARPAS cell Line source: Dr Abraham Karpas at the University of Cambridge.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 5

Flow cytometric analysis of untreated Jurkat cells, using Bcl-xL (54H6) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 6

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human A1/Bfl-1 protein (hA1/Bfl-1; +), using A1/Bfl-1 (D1A1C) Rabbit mAb.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 7

Western blot analysis of extracts from A673 and C2C12 cell lines, using Bcl-w (31H4) Rabbit mAb.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 8

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.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 9

Western blot analysis of extracts from various cell lines using Bcl-2 (D55G8) Rabbit mAb (Human Specific).

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 10

Western blot analysis of extracts from Jurkat cells, untreated or treated with paclitaxel (1 μM, overnight) and with or without λ phosphatase, using Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb (upper) or Bcl-2 #2876 (lower).

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 11

Flow cytometric analysis of L-929 cells using Mcl-1 (D2W9E) Rabbit mAb (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was use as a secondary antibody.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 12

Western blot analysis of extracts from KARPAS-299 and Raji cell lines, untreated (-) or treated with TPA #4174 (200 nM, 30 min; +), using Phospho-Mcl-1 (Thr163) (D5M9D) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). KARPAS cell Line source: Dr Abraham Karpas at the University of Cambridge.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 13

Confocal immunofluorescent analysis of HeLa cells using Bcl-xL (54H6) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 14

Western blot analysis of extracts from U-937 and UACC-62 cells using A1/Bfl-1 (D1A1C) Rabbit mAb.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 15

Western blot analysis of recombinant Bcl-w (amino acids 1-172) using Bcl-w (31H4) Rabbit mAb.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 16

Confocal immunofluorescent analysis of MCF7 (left), L-929 (center), and SK-OV-3 (right) cells using Mcl-1 (D2W9E) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 17

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Bcl-xL (54H6) Rabbit mAb.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 18

Immunoprecipitation of Mcl-1 from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Mcl-1 (D2W9E) Rabbit mAb. Western blot analysis was performed using Mcl-1 (D2W9E) Rabbit mAb. A conformation-specific secondary antibody was used to avoid cross-reactivity with IgG.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 19

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Bcl-xL (54H6) Rabbit mAb in the presence of control peptide (left) or Bcl-xL Blocking Peptide #1225 (right).

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 20

Western blot analysis of extracts from 293T cells, mock transfected or transfected with a construct expressing full-length human Mcl-1 (hMcl-1; +) or mouse Mcl-1 (mMcl-1; +), using Mcl-1 (D2W9E) Rabbit mAb.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 21

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, showing cytoplasmic localization, using Bcl-xL (54H6) Rabbit mAb.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 22

Western blot analysis of extracts from various cell lines using Mcl-1 (D2W9E) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 23

Immunoprecipitation of Bcl-xL from Jurkat cell extracts, using Bcl-xL (54H6) Rabbit mAb. Lane 1 is the lysate control, lane 2 is antibody alone and lane 3 is antibody plus lysate.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 24

Western blot analysis of extracts from Jurkat and HeLa (human), COS (monkey), NIH/3T3 and L929 (mouse), and PC12 and C6 (rat) cells, using Bcl-xL (54H6) Rabbit mAb.

Pro-Survival Bcl-2 Family Antibody Sampler Kit II: Image 25

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® BcL-xL siRNA I (+) or SignalSilence® Bcl-xL siRNA II #6363 (+), using Bcl-xL (54H6) Rabbit mAb #2764 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Bcl-xL (54H6) Rabbit mAb confirms silencing of Bcl-xL expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

To Purchase # 17229T
Product # Size Price
17229T
1 Kit  (7 x 20 µl) N/A

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Bcl-2 (D55G8) Rabbit mAb (Human Specific) 4223 20 µl
  • WB
  • IP
H 26 Rabbit IgG
Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb 2827 20 µl
  • WB
  • F
H 28 Rabbit IgG
Mcl-1 (D2W9E) Rabbit mAb 94296 20 µl
  • WB
  • IP
  • IF
  • F
H M R 40 (human), 35 (rodent) Rabbit IgG
Phospho-Mcl-1 (Thr163) (D5M9D) Rabbit mAb 14765 20 µl
  • WB
  • IP
H 40 Rabbit IgG
Bcl-xL (54H6) Rabbit mAb 2764 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H Mk M R 30 Rabbit IgG
A1/Bfl-1 (D1A1C) Rabbit mAb 14093 20 µl
  • WB
  • IP
H 18 Rabbit IgG
Bcl-w (31H4) Rabbit mAb 2724 20 µl
  • WB
H M R 18 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Pro-Survival Bcl-2 Family Antibody Sampler Kit II provides an economical means to examine several members of the Bcl-2 family. The kit contains enough primary antibody to perform two western blot experiments.

Specificity / Sensitivity

Each antibody in the Pro-Survival Bcl-2 Family Antibody Sampler Kit II recognizes endogenous levels of its specific target. The antibodies do not cross-react with other Bcl-2 family members. A1/Bfl-1 (D1A1C) Rabbit mAb may cross-react with an unknown protein at 50 and 130 kDa in some cell lines. Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb detects endogenous of human Bcl-2 only when phosphorylated at Ser70. Phospho-Mcl-1 (Thr163) (D5M9D) Rabbit mAb recongizes endogenous levels of Mcl-1 only when phosphorylated at Thr163. This antibody may also cross-react with an unidentified protein at 70 kDa in some cell lines.

Source / Purification

Rabbit monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Gly47 of human Bcl-2, Asp61 of human Bcl-xL, Pro60 of mouse Mcl-1, Gly29 of human A1/Bfl-1, and Ala39 of human Bcl-w. Phospho-specific rabbit monoclonal antibodies are produced by immunizing animlars with synthetic phospho-peptides correspoding to residues surrounding Ser70 of human Bcl-2 and Thr163 of human Mcl-1.

Background

The Bcl-2 family consists of a number of evolutionarily conserved proteins containing Bcl-2 homology domains (BH) that regulate apoptosis through control of mitochondrial membrane permeability and release of cytochrome c (1-3). Four BH domains have been identified (BH1-4) that mediate protein interactions. The family can be separated into three groups based upon function and sequence homology: pro-survival members include Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w; pro-apoptotic proteins include Bax, Bak and Bok; and "BH3 only" proteins Bad, Bik, Bid, Puma, Bim, Bmf, Noxa and Hrk. Interactions between death-promoting and death-suppressing Bcl-2 family members has led to a rheostat model in which the ratio of pro-apoptotic and anti-apoptotic proteins controls cell fate (4). Thus, pro-survival members exert their behavior by binding to and antagonizing death-promoting members. In general, the "BH3-only members" can bind to and antagonize the pro-survival proteins leading to increased apoptosis (5). While some redundancy of this system likely exists, tissue specificity, transcriptional and post-translational regulation of many of these family members can account for distinct physiological roles.

Several phosphorylation sites have been identified within Bcl-2 including Thr56, Ser70, Thr74 and Ser87 (6). These phosphorylation sites may be targets of the ASK1/MKK7/JNK1 pathway, and phosphorylation of Bcl-2 may be a marker for mitotic events (7,8). Mutation of Bcl-2 at Thr56 or Ser87 inhibits its anti-apoptotic activity during glucocorticoid-induced apoptosis of T lymphocytes (9). Interleukin 3 and JNK-induced Bcl-2 phosphorylation at Ser70 may be required for its enhanced antiapoptotic functions (10).

Mcl-1 is phosphorylated in response to treatment with phorbol ester, microtubule-damaging agents, oxidative stress, and cytokine withdrawal (11-14). Phosphorylation at Thr163, the conserved MAP kinase/ERK site located within the PEST region, slows Mcl-1 protein turnover (13) but may prime the GSK-3 mediated phosphorylation at Ser159 that leads to Mcl-1 destabilization (14).

  1. Cory, S. et al. (2003) Oncogene 22, 8590-607.
  2. Antonsson, B. and Martinou, J.C. (2000) Exp Cell Res 256, 50-7.
  3. Sharpe, J.C. et al. (2004) Biochim Biophys Acta 1644, 107-13.
  4. Korsmeyer, S.J. et al. (1993) Semin Cancer Biol 4, 327-32.
  5. Bouillet, P. and Strasser, A. (2002) J Cell Sci 115, 1567-74.
  6. Maundrell, K. et al. (1997) J Biol Chem 272, 25238-42.
  7. Yamamoto, K. et al. (1999) Mol Cell Biol 19, 8469-78.
  8. Ling, Y.H. et al. (1998) J Biol Chem 273, 18984-91.
  9. Huang, S.T. and Cidlowski, J.A. (2002) FASEB J 16, 825-32.
  10. Deng, X. et al. (2001) J Biol Chem 276, 23681-8.
  11. Domina, A.M. et al. (2000) J Biol Chem 275, 21688-94.
  12. Inoshita, S. et al. (2002) J Biol Chem 277, 43730-4.
  13. Domina, A.M. et al. (2004) Oncogene 23, 5301-15.
  14. Maurer, U. et al. (2006) Mol Cell 21, 749-60.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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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's 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's 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.

For Research Use Only. Not For Use In Diagnostic Procedures.
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