APPLICATION FOCUS. Application Solutions for Western Blotting

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1 APPLICATION FOCUS Application Solutions for Western Blotting

2 WESTERN BLOTTING Companion Products Solutions for consistently better blotting. Companion products from Cell Signaling Technology (CST) are the same reagents used by our scientists when validating our primary antibodies, so we have the experience to support every step of your western blot (WB) or immunoprecipitation (IP) experiment. These products are thoroughly validated and work optimally with our primary antibodies and protocols, so you obtain clean blots with specific signal, for reproducible and publishable results in the least amount of time. { } Western blotting success is dependent on the quality of your antibodies and your protocol. CST provides extensive western blotting reference information from our rigorous in-house testing to support your experiments. Save time by skipping antibody titrations, because optimal dilutions and dilution buffers are identified for every CST antibody. These specifications are generated and supported by testing and are provided on the datasheet that accompanies every antibody. CST Technical Support At CST, providing exceptional customer service and technical support is a top priority. Our scientists work at the bench daily to produce and validate our antibodies, so they have hands-on experience and in-depth knowledge of each antibody s performance. In the process, these same scientists generate valuable reference information that they use to answer your questions and help troubleshoot your experiment by phone or . (USA & Europe) (China) (Japan) Western Blotting Application Solutions Kit #12957 It takes multiple buffers and reagents to complete your western blot experiment. Save your valuable time by choosing the convenience of the CST Western Blotting Application Solutions Kit, which contains pre-made buffers and reagents to support each step of your protocol. KIT INCLUDES #9 Cell Lysis Buffer (1X) #7722 Blue Loading Buffer Pack #772 Prestained Protein Marker, Broad Range (Premixed Format) #9998 BSA #774 Anti-rabbit IgG, HRP-linked Antibody #77 Anti-mouse IgG, HRP-linked Antibody #855 PMSF #4 Tris-Glycine SDS Running Buffer (1X) #129 Nitrocellulose Sandwiches #1259 Tris-Glycine Transfer Buffer (1X) Tween 2 (TBST-1X) #88 SignalFire ECL Reagent % SDS-PAGE Prestained Protein Marker, Broad Range (Premixed Format) #772: WB separated by 1-2% SDS-PAGE.

3 How important are protease and phosphatase inhibitors? You Decide. It takes time and resources to obtain high quality cell extracts for experiments. Protect the integrity of your samples by always including protease and phosphatase inhibitors in your lysis buffer. Protease and Phosphatase Inhibitor Cocktails offer the convenience of broad spectrum enzymatic inhibition at an easy to use 1X concentration. PROTEASE AND PHOSPHATASE INHIBITORS #5872 #5871 #587 #855 Phosphatase Inhibitors preserve phosphorylation Protease/Phosphatase Inhibitor Cocktail (1X) #5872: WB analysis of extracts from NIH/T cells, serum-starved overnight and treated with hpdgf-bb #8912 (1 ng/ml, 5 min), prepared in lysis buffer in the absence of phosphatase inhibitors (left) or with #5872 added (right) and was incubated at 7 C for the indicated time following cell lysis, using Phospho-Akt (Ser47) (D9E) XP Rabbit mab #4 (upper) or Akt (pan) (C7E7) Rabbit mab #491 (lower). In the absence of phosphatase inhibitors, phospho-akt signal drops significantly after hr, demonstrating rapid loss of phosphorylation at later time points. no phosphatase inhibitors #5872 added 2 1 Phospho-Akt (Ser 47) 4 Protease/Phosphatase Inhibitor Cocktail (1X) Protease Inhibitor Cocktail (1X) Phosphatase Inhibitor Cocktail (1X) PMSF In the presence of the phosphatase inhibitor cocktail, phospho-akt signal is preserved through all monitored time points. Akt 2 2 hrs Protease Inhibitors prevent degradation Protease/Phosphatase Inhibitor Cocktail (1X) #5872: WB analysis of NIH/T cell extracts prepared in lysis buffer in the absence of protease inhibitors (left) or with #5872 added (right) was performed at the indicated time points following cell lysis and storage on ice, using β-catenin (D1A8) XP Rabbit mab #84. In the absence of protease inhibitors, β-catenin signal fades within hr after lysis, indicating protein degradation. no protease inhibitors #5872 added 2 1 β-catenin degradation product 2 2 hrs In the presence of the protease inhibitor cocktail, β-catenin degradation is slowed significantly and signal is still present 2 hr after lysis.

4 Reliable reagents to support your protocol SDS-PAGE #4 Tris-Glycine SDS Running Buffer (1X) #7722 Blue Loading Buffer Pack #7727 Biotinylated Protein Ladder Detection Pack WET TRANSFER #1259 Tris-Glycine Transfer Buffer (1X) #129 Nitrocellulose Sandwiches BLOCKING PRIMARY Ab INCUBATION #9998 BSA tissue or cells homogenous solution SAMPLE PREPARATION #77 BCA Protein Assay Kit *#9 Cell Lysis Buffer (1X) All CST lysis buffers contain protease and phosphatase inhibitors. We recommend adding 1 mm PMSF immediately before use. TIP: Use #77 with #9 to reliably quantify the protein concentration of your samples for more accurate loading. (9 2 ) #772 Prestained Protein Marker, Broad Range (Premixed Format) (7 175 ) Cell Lysis Buffer (1X) #9 Blue Loading Buffer Pack #7722 is ideal for lysate concentration determination and IP experiments because it lyses cells under non-denaturing conditions. allows easy observation of sample progression through the gel matrix. Blue Loading Buffer Pack contains X SDS Loading Buffer and X DTT reducing agent for use in SDS-PAGE analysis. IP from transfected 29T cells l tro ut con inp % 1 # #1 1 His-Tag IgG Cell Lysis Buffer (1X) #9: IP of His-Tag protein from transfected 29T cells using Rabbit (DA1E) mab IgG XP Isotype Control #9 (lane 2) or His-Tag (DI1O) XP Rabbit mab #1298 (lane ). Lane 1 is 1% input. WB analysis was performed using #1298. Cell lysates were prepared using # Nitrocellulose Sandwiches #129 provide a convenient alternative to cutting membrane and filter paper by hand and are suitable for most common transfer methods including tank, semi-dry, and vacuum blotting. These membrane sandwiches consist of a 7 x 8.5 cm precut.2 μm pore size nitrocellulose membrane and 2 sheets of.4 mm thick blotting paper. Western blotting tools for success White paper Helpful tips and explanations to support your western blotting experiment are available in our white paper A Guide to Successful Western Blotting, request a copy at Protocol guide Protocols optimized by our scientists and used to validate each antibody are available, so you spend less time troubleshooting. To request a copy of our Protocols & Troubleshooting Guide visit

5 WASHING SECONDARY Ab INCUBATION #77 Anti-mouse IgG, HRP-linked Antibody #774 Anti-rabbit IgG, HRP-linked Antibody WASHING DETECTION #88 SignalFire ECL Reagent #12 SignalFire Plus ECL Reagent #12757 SignalFire Elite ECL Reagent #5571 Anti-biotin (D5A7) Rabbit mab (HRP Conjugate) #775 Anti-biotin, HRP-linked Antibody IMAGING Anti-rabbit IgG, HRP-linked Antibody #774: WB analysis of extracts from human non-small cell lung carcinoma cell lines NCI-H2228 and NCIH122, using ALK (D5F) XP Rabbit mab # and #774 (incubated for 1 hr at room temperature). Variants denoting fusions of different EML4 exons (v1 or v) are indicated. Controls table Low background detection of splice variants I-H CI-H C N N 2 1 EML4ALK v1 EML4ALK v provides a more robust signal and extended duration of signal output than other luminolbased systems. SignalFire ECL Reagent is an enhanced luminol-based system and capable of detecting low picogram amounts protein. SignalFire ECL Reagent #88: WB analysis of recombinant human HO-1 (hho-1) using HO-1 (DG11) Rabbit mab #585 and chemiluninescent detection reagent #88. Titration to picogram levels sec 1 min min 5 min 1 ng 5 ng 2.5 ng 1 ng pg 2 pg 1 pg pg 25 pg 1 pg 5 pg 2.5 pg yields high sensitivity with short reaction times. Designed for use with rabbit polyclonal and monoclonal antibodies, this affinity purified goat anti-rabbit IgG (heavy and light chain) antibody is conjugated to horseradish peroxidase for chemiluminescent detection. SignalFire ECL Reagent #88 Exp. Time Anti-rabbit IgG, HRP-linked Antibody #774 hho-1 4 CST has compiled a table of positive and negative controls for many of our antibody targets, so you can be confident your protocol and reagents are working as expected. Visit to view our online controls table so you can make your own control extracts. Videos CST scientists perform about one thousand western blots daily to validate our existing and new antibodies. Go to to watch our western blotting protocol and troubleshooting guide videos.

6 UNITED STATES Hours: M F 8:am to 8:pm [EST] Orders: Support: CHINA Tel: Support (China): /GreatQ Support (Asia Pacific): EUROPE, MIDDLE EAST & AFRICA Tel: +1 () JAPAN Tel: Support: AUTHORIZED DISTRIBUTORS See a full list of our distributors online. CST Antibody Performance Guarantee To learn more, please visit: FRONT COVER PHOTO: Andreas Nelsbach, Senior Product Scientist (left), has been with CST for over 14 years and Katrina Peterson, Production Associate (right), has been with CST for over years. FOUNDED BY RESEARCH SCIENTISTS IN 1999, Cell Signaling Technology (CST) is a private, familyowned company with over 4 employees worldwide. Active in the field of applied systems biology research, particularly as it relates to cancer, CST understands the importance of using antibodies with high levels of specificity and lot-to-lot consistency. It s why we produce all of our antibodies in house, and perform painstaking validations for multiple applications. And the same CST scientists who produce our antibodies also provide technical support for customers, helping them design experiments, troubleshoot, and achieve reliable results. We do this because that s what we d want if we were in the lab. Because, actually, we are. CST Product Scientists, Beverly, MA USA Printed on recycled paper (25% post-consumer waste fiber) using vegetable inks and processed chlorine free. 1/21 Cell Signaling Technology, Inc. Cell Signaling Technology, CST, SignalFire, and XP are trademarks of Cell Signaling Technology, Inc. Tween 2 is a registered trademark of ICI Americas, Inc. These antibodies are available exclusively from Cell Signaling Technology (CST) and are not available from other vendors. Select rabbit monoclonal antibodies are developed, validated, and produced at CST using in part technology under license (granting certain rights including those under U.S. Patents No. 5,75,) from Epitomics, Inc. CPI1P For Research Use Only. Not For Use in Diagnostic Procedures.

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