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

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

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Western blot analysis of HeLa cell extracts, untreated (-) or Bcl-2 knock-out (+), using Bcl-2 (D55G8) Rabbit mAb #4223 (upper), or β-actin (13E5) Rabbit mAb #4970 (lower).

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).

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.

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.

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.

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.

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

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.

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

Flow cytometric analysis of Jurkat cells, using Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb versus propidium iodide (DNA content).

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

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.

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.

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

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

Confocal immunofluorescent analysis of SH-SY5Y cells using Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb (red) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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.

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.

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.

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

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.

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).

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).

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

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

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

To Purchase # 17229T
Product # Size Price
17229T
1 Kit  (7 x 20 µl) $ 485

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
  • IF
  • 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 M R Mk 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.

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

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