Revision 1

#78551Store at -20C

1 Kit

(6 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
p16 INK4A (E5F3Y) Rabbit mAb 29271 20 µl 16 kDa Rabbit IgG
p21 Waf1/Cip1 (E2R7A) Rabbit mAb 37543 20 µl 21 kDa Rabbit IgG
Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb 9718 20 µl 15 kDa Rabbit IgG
Lamin B1 (E6M5T) Rabbit mAb 17416 20 µl 68, 45 kDa Rabbit IgG
HMGB1 (D3E5) Rabbit mAb 6893 20 µl 29 kDa Rabbit IgG
IL-6 (D5W4V) XP® Rabbit mAb 12912 20 µl 24 kDa Rabbit IgG
TNF-α (D2D4) XP® Rabbit mAb 11948 20 µl 17,25,28 kDa Rabbit IgG
MMP-2 (D2O4T) Rabbit mAb 87809 20 µl 64,72 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

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

Description

The Mouse Reactive Senescence Marker Antibody Sampler Kit provides an economical means of detecting multiple markers of cellular senescence in mouse samples. The kit includes enough antibodies to perform two western 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 antibodies.

Background

Senescence is characterized by stable stress-induced proliferative arrest and resistance to mitogenic stimuli, as well as the secretion of proteins such as cytokines, growth factors, and proteases. These secreted proteins comprise the senescence-associated secretory phenotype (SASP). Senescent cells are thought to accumulate as an organism ages, and contribute to age-related diseases, including cancer, through promotion of inflammation and disruption of normal cellular function (1,2).
Because there is no single biomarker that can be used to definitively identify senescent cells, researchers must rely on a collection of biomarkers commonly associated with senescence (3). The Mouse Reactive Senescence Marker Antibody Sampler Kit provides a collection of antibodies to commonly used biomarkers of senescence-associated cell cycle arrest (p16 INK4A, p21 Waf1/Cip1), senescence-associated DNA damage (gamma-Histone H2A.X), and the SASP (HMGB1, IL-6, TNF-α, MMP2). The kit also includes an antibody to lamin B1, which is frequently reduced in senescent cells.

  1. Tchkonia, T. et al. (2013) J Clin Invest 123, 966-72.
  2. Sun, Y. et al. (2018) Trends Mol Med 24, 871-885.
  3. Gorgoulis, V. et al. (2019) Cell 179, 813-827.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered 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

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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 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.

    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
    #78551

    Mouse Reactive Senescence Marker Antibody Sampler Kit

    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 1 Expand Image

    Simple Western™ analysis of lysates (1.0 mg/mL) from 3T3-L1 cells using MMP-2 (D2O4T) Rabbit mAb #87809. The virtual lane view (left) shows the target band (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.

    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from Raw 264.7 cells, untreated (-) or treated (+) with LPS (100 ng/mL, 6 hr) and Brefeldin A #9972 (300 ng/mL, last 3 hr of stimulation), using TNF-α (D2D4) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from Raw 264.7 cells, untreated (-) or treated with LPS (100 ng/ml, 6 hr) and Brefeldin A #9972 (300 ng/ml, last 3 hr of stimulation; +), using IL-6 (D5W4V) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines and rat spleen tissue using Lamin B1 (E6M5T) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Relatively low expression of Lamin B1 protein in U-87 MG cells is consistent with the predicted expression pattern.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 5 Expand Image
    Immunoprecipitation of p16 INK4A protein from mouse A20 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is p16 INK4A (E5F3Y) Rabbit mAb. Western blot analysis was performed using p16 INK4A (E5F3Y) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody. 
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing full-length mouse p16 INK4A protein (mINK4A-Myc/DDK; +) or full-length mouse p15 INK4B protein (mINK4B-Myc/DDK; +), using p16 INK4A (E5F3Y) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from MEF cells, untreated (-) or treated with Doxorubicin #5927 (250 nM, 24 hours followed by 7 days without doxorubicin; +), using p16 INK4A (E5F3Y) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). p16 INK4A protein expression is induced by doxorubicin as expected.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from various mouse cell lines using p16 INK4A (E5F3Y) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Negative expression of p16 INK4A protein in NIH/3T3 cells is consistent with the predicted expression pattern.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from control C2C12 cells (lane 1) or p21 Waf1/Cip1 knockout C2C12 cells (lane 2) using p21 Waf1/Cip1 (E2R7A) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the p21 Waf1/Cip1 knockout C2C12 cells confirms specificity of the antibody for p21 Waf1/Cip1.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using HMGB1 (D3E5) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 11 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.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from U87, 3T3-L1 and C2C12 cells using MMP-2 (D4O4T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from untreated or UV-treated 293 cells, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (upper) or Histone H2A.X Antibody #2595 (lower).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ovarian clear cell carcinoma using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb performed on the Leica BOND Rx.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of 1 ng recombinant mouse TNF-α #5178 using TNF-α (D2D4) XP® Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of recombinant Mouse His6Interleukin-6 (mHis6IL-6) using IL-6 (D5W4V) XP® Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human breast using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 18 Expand Image
    Western blot analysis of extracts from C2C12, NIH/3T3 (mouse), and MCF7 (human) cells, untreated (-) or treated with Nutlin 3a (10 μM, 24 hr; +), using p21 Waf1/Cip1 (E2R7A) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse lung using HMGB1 (D3E5) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 20 Expand Image
    Western blot analysis of extracts from the media of mouse bone marrow derived macrophages (mBMDM), untreated (-) or treated with Lipopolysaccharides (LPS) #14011 (50 ng/ml, 4 hr; +) followed by Nigericin (15 μM, 45 min; +), using HMGB1 (D3E5) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 21 Expand Image
    Immunoprecipitation of MMP-2 protein from U87 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is MMP-2 (D2O4T) Rabbit mAb. Western blot analysis was performed using MMP-2 (D2O4T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb performed on the Leica BOND Rx.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 23 Expand Image
    Immunoprecipitation of TNF-α from Raw 264.7 cell extracts, treated with LPS (100 ng/mL, 6 hr) and Brefeldin A #9972 (300 ng/mL, last 3 hr of stimulation), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or TNF-α (D2D4) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using TNF-α (D2D4) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) #3678 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as sencondary antibodies.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 24 Expand Image
    Immunoprecipitation of IL-6 from Raw 264.7 cell extracts treated with LPS (100 ng/ml, 6 hr) and Brefeldin A #9972 (300 ng/ml, last 3 hr of stimulation) using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or IL-6 (D5W4V) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using IL-6 (D5W4V) XP® Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded BeWo cell pellet (left, high-expressing) or U-87 MG cell pellet (right, low-expressing) using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 26 Expand Image
    Immunoprecipitation of p21 Waf1/Cip1 protein from extracts of C2C12 cells treated with Nutlin 3a (10 μM, 24 hr).  Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is p21 Waf1/Cip1 (E2R7A) Rabbit mAb. Western blot analysis was performed using  p21 Waf1/Cip1 (E2R7A) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody. 
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcioma using HMGB1 (D3E5) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb, showing nuclear localization.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 29 Expand Image
    Confocal immunofluorescent analysis of Raw 264.7 cells, treated with LPS (100 ng/mL, 6 hr; left) or untreated (right), using TNF-α (D2D4) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 30 Expand Image
    Confocal immunofluorescent analysis of Raw 264.7 cells, untreated (left) or treated with LPS (100 ng/ml, 6 hr; right), using IL-6 (D5W4V) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded human esophageal adenocarcinoma using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcioma using HMGB1 (D3E5) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 33 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 34 Expand Image
    Flow cytometric analysis of Raw 264.7 cells, untreated (blue) or treated with LPS (100 ng/ml, 6 hr) and Brefeldin A #9972 (300 ng/ml, last 3 hr of treatment) (green), using TNF-α (D2D4) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 35 Expand Image
    Flow cytometric analysis of Raw 264.7 cells, treated with LPS (100 ng/ml, 6 hr) and Brefeldin A #9972 (300 ng/ml, last 3 hr of LPS treatment) (green) or Brefeldin A alone (300 ng/ml, 3 hr) (blue) using IL-6 (D5W4V) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded human squamous cell carcinoma of the tonsil using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 37 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate carcioma using HMGB1 (D3E5) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 38 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma untreated (left) or lambda-phosphatase-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 39 Expand Image
    Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 40 Expand Image
    Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 41 Expand Image
    Immunohistochemical analysis of paraffin-embedded HT-29 cells untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 42 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 43 Expand Image
    Immunohistochemical analysis of paraffin-embedded human mature ovarian teratoma using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 44 Expand Image
    Flow cytometric analysis of HeLa cells, untreated (blue) or treated with UV (100 mJ, 2hr recovery; green) using Phospho-H2A.X (Ser139) (20E3) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® isotype control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 45 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ovarian clear cell carcinoma using Lamin B1 (E6M5T) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 46 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human pancreas using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 47 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human prostate using Lamin B1 (E6M5T) Rabbit mAb.
    Mouse Reactive Senescence Marker Antibody Sampler Kit: Image 48 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human thymus using Lamin B1 (E6M5T) Rabbit mAb.
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    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.