SNAP i.d. Protein Detection System. Superior Blots in a Snap!

Transcription

1 SNAP i.d. Protein Detection System Superior Blots in a Snap!

2 SNAP i.d. Protein Detection System Superior Blots in a Snap! The SNAP i.d. Protein Detection System revolutionizes immunodetection every time in record time! Unlike conventional western blotting, where diffusion is the primary means of reagent transport, the SNAP i.d. system applies a vacuum to actively drive reagents through the membrane. This novel method allows you to optimize your blotting conditions in record time for maximum results. The SNAP i.d. system minimizes overblocking by using lower concentrations of blocking agents. In addition, more effective wash steps remove unwanted contaminants from the membrane. With the SNAP i.d. system, you ll achieve incredibly low background, high signal-to-noise ratios, reproducibility from blot to blot, and sensitivities that are the same or better than traditional immunodetection techniques. And did we mention that your immunodetection time will drop from 4 hours to only 30 minutes? 45 min h h 1 h 3 h 15 min SNAP i.d. in the Western Blotting Workflow 2

3 Advantages of the SNAP i.d. System Dynamic vacuum actively drives reagents through blotting membrane. Quality equal or better signal-to-noise ratios than standard western blotting. Fast reduces immunodetection time from 4 hours to 30 minutes. Simple incorporates blocking, washing, and antibody incubation steps. Compatible works with standard gel sizes and protocols. Efficient optimizes your protocol with a new antibody in 30 minutes. Blot holders accept three common sizes: 7.5 x 8.8 cm, 4.6 x 8.8 cm and 3.2 x 8.8 cm Two blot holders can be run independently or in parallel; up to 6 blots can be processed simultaneously for fast optimization of protocols. Integrated flow distributors drive reagent flow, reducing immunodetection time and ensuring consistent results. Antibody collection tray collects primary antibodies for reuse. The SNAP i.d. System is validated for chemiluminescent and fluorescence detection for compatibility with existing western blotting protocols. 3

4 Standard western blotting protocol vs. SNAP i.d. system Optimize in a SNAP! Use the same amount of antibody. Don t compromise on background signals. Standard Western Blot SNAP i.d. protein detection system Blocking Step Time 1 h 20 s Concentration 5% NFDM 0.5% NFDM Reagent transport Diffusion Actively-driven through membrane Primary Antibody Step Time 1 18 h 10 min Concentration 1x 3x in 1/3 volume (= same quantity) Reagent transport Diffusion Actively-driven through membrane Washing (3x) step Time 3 x 5 min = 15 min 3 x 20 s = 1 min Concentration 1x 1x Reagent transport Diffusion Actively-driven through membrane Secondary Antibody step Washing (3x) step Time 1 h 10 min Time 3 x 5 min = 15 min 3 x 20 s = 1 min Total Time 4 20 h 22 min 4

5 Blocking buffers are used at lower concentrations with the SNAP i.d. system. Our proprietary blot holders and actively-driven reagent process ensure that the pores of the membrane are adequately blocked. Likewise, wash steps actively flush the membrane instead of just rinsing the surface. The design of the SNAP i.d. system requires that antibodies be used at three times the normal concentration but in only one third of the volume, so you can get the same or better results with the same amount of antibody! Standard Western Blot 5% NFDM A Improve your signal! Use low concentrations of blocking reagents with the SNAP i.d. system to improve quality. SNAP i.d. Protein Detection System 0.5% NFDM B % NFDM C % NFDM D Non-Fat Dry Milk (NFDM) is an efficient blocking solution commonly used in western blotting; however, its high blocking capacity may compromise the protein signal. To demonstrate this, a two-fold dilution series of rat liver lysate (12 µg in lane 1 to 0.09 µg in lane 8) was resolved with SDS-PAGE prior to blotting and immunodetection. (The primary antibody was mouse anti-glyceraldehyde-3-phosphate Dehydrogenase (GAPDH); the secondary antibody was HRP-conjugated goat anti-mouse). Blot A used a standard immunodetection protocol (block for 1 hour in 5% NFDM, incubate in primary (1:40,000) or secondary antibody (1:50,000) for one hour, wash three times following incubations). Blot B, C and D were assembled in SNAP i.d blot holders and blocked for 20 seconds with either 0.5, 0.1 or 0.05% NFDM respectively. The blots were incubated for 10 minutes with anti-gapdh (1:13,000), washed immediately and incubated for 10 minutes with HRP goat anti-mouse (1:10,000). Results show an increase in sensitivity with a decrease in milk concentration. 5

6 The SNAP i.d. system is compatible with your favorite membrane and detection method! The SNAP i.d. system achieves high quality results without any additional reagent consumption (e.g., antigen, antibody or detection reagents). The system s unique design enables the use of small volumes for antibody incubations with either polyvinylidene difluoride (PVDF) or nitrocellulose blotting membranes. Because it uses a vacuum to actively drive reagents through the membrane, blocking and washing steps are achieved thoroughly and rapidly. The SNAP i.d. system is compatible with fluorescent, chemiluminescent or chromagenic detection methods. Moreover, the sequence of steps required to process a western blot with the SNAP i.d. system is identical to that used in traditional immunodetection. Any Membrane HSP70 70 Immobilon -P Membrane Immobilon-P SQ Membrane Immobilon-FL Membrane Nitrocellulose Membrane Any Detection Method HSP70 70 HRP AP Chromogenic Fluorescent 6

7 Low, Medium or Highly Expressed Proteins Albumin Transferrin Adenovirus Hexon Cyclophilin MAP Kinase ErK Nestin EGF Receptor Vimentin Akt-1 P-53 Oncoprotein A variety of cell lysates from rat liver, cancer or stem cells, and human serum, were resolved by SDS-PAGE, transferred to Immobilon-P membrane and subjected to immunodetection according to the SNAP i.d. protocol. Proteins of different molecular weight and abundance were visualized after incubating the blots with Immobilon Western HRP substrate and exposing to x-ray film for one to five minutes. 7

8 The SNAP i.d. system is compatible with a wide variety of antibodies. Here are a few we ve tested. Type of Antibody Host Antibody Name Catalogue No. Sample Type Primary Antibodies Monoclonal Primary Antibodies Polyclonal Secondary Antibodies Mouse Anti-GAPDH MAB374 Rat liver Mouse Anti-Actin, clone C4 MAB1501 Rat liver Mouse Anti-His tag, clone 4D E. coli with His-tag Mouse Anti-Akt, clone SKB A431 cells Mouse Anti-Adenovirus MAB8052 HEK infected Mouse Anti-Neuronal Nuclei (NeuN) MAB377 Rat brain Mouse Anti-Synaptophysin MAB368 Human brain Mouse Anti-PP2A, C subunit, clone 1D A431 and HeLa cells Mouse Anti-p38, SAPK2, clone 2F A431 cells Mouse Anti-FAK, clone Jurkat cells Mouse Anti-CDK2, clone AN HeLa cells Mouse Anti-Tubulin β III CBL412 Ren Vx cells Mouse Anti-Neurofilament 70, clone DA2 MAB1615 Ren Vx cells Goat Anti-Vimentin AB1620 ReNcell CX lysates Rabbit Anti-p53 AB565 Human serum Rabbit Anti-MAP Kinase 1/2 (Erk-1/2) MDCK cells Rabbit Anti-Nestin AB5922 ReNcell CX lysates Rabbit Anti-β-Catenin A431 cells Rabbit Anti-Cyclophilin A A431 cells Rabbit Anti-CREB HeLa cells Rabbit Anti-phospho-IKKα (Ser. 176/Ser. 180) Jurkat cells Rabbit Anti-Caspase Jurkat cells Rabbit Anti-Sox2 AB5603 Mouse embryonic stem cell Sheep Anti-Transferrin AB7142 Human serum Goat Anti-mouse IgG, HRP conjugate AP124P Goat Anti-rabbit IgG, HRP conjugate AP132P Goat Anti-mouse IgG, Cy5 conjugate AP200S Rabbit Anti-sheep IgG, HRP conjugate AP147P Rabbit Anti-goat IgG, HRP conjugate AP106P *To order these antibody part numbers from Fisher Scientific please add an MI to the end of each Millipore catalogue number above. 8

9 Ordering Information SNAP i.d. Protein Detection System Product Description Quantity Catalogue No. SNAP i.d. Protein Detection System SNAP i.d. Consumables and Accessories WBAVDBASE Single Blot Holder 30/pk WBAVDBH01 Double Blot Holder 30/pk WBAVDBH02 Triple Blot Holder 20/pk WBAVDBH03 Antibody Collection Tray 20/pk WBAVDABTR SNAP i.d. Blot Roller WBAVDR0LL Related Products Immobilon-P Transfer Membrane, 7 x 8.4 cm 50 sheets IPVH07850 Immobilon-P Transfer Membrane, 26.5 x 375 cm 1 roll IPVH00010 Immobilon-P SQ Transfer Membrane, 7 x 8.4 cm 50 sheets ISEQ07850 Immobilon-P SQ Transfer Membrane, 26.5 x 375 cm 1 roll ISEQ00010 Immobilon-FL Transfer Membrane, 26.5 x 375 cm 1 roll IPFL00010 Immobilon-P Blotting Sandwich, 7 x 8.4 cm 20 sandwiches IPSN07852 Immobilon Blotting Filter Paper, 7 x 8.4 cm 100 sheets IBFP0785C Immobilon Western Chemiluminescent HRP Substrate 50 ml WBKLS ml WBKLS ml WBKLS0500 Visualizer Spray & Glow ECL Western Blotting Detection System ReBlot Plus Kit 2500 ReBlot Plus Mild Antibody Stripping Solution, 10x 2502 ReBlot Plus Strong Antibody Stripping Solution, 10x 2504 Chemical Duty Pump, 115 V/60 Hz Chemical Duty Pump, 220 V/50 Hz Vacuum Filtering Flask, 1 L Silicone No. 8 Perforated Stopper, 5/pk Millipore Forceps SS WP WP XX XX XX Contact your Millipore sales representative for more information about the SNAP i.d. system. 9

10 Millipore has your complete solution for immunodetection and western blotting. Sample preparation Whether you are lysing cells, extracting proteins, filtering samples, or performing immunoprecipitation (IP), check out our sample preparation tools. You ll find Amicon Ultra protein concentrators, pre-made cell lysates, and Catch & Release IP kits for simplifying your protein research. Blotting membranes For superior blots, use Immobilon PVDF transfer membranes. We offer several varieties, including formats for low molecular weight proteins and fluorescent detection. Our pre-cut blotting sandwiches ensure even faster processing! Antibodies Primary: Millipore offers thousands of high quality, validated antibodies from the expertise of Chemicon and Upstate. Focus areas include neuroscience, cell signaling, apoptosis, cellular adhesion and structure, nuclear function, and protein tags. Secondary: Millipore provides secondary antibodies with a variety of host species and conjugates for both enzymatic and fluorescent detection. Special formats, including F(ab ) 2 fragment and species-absorbed, are also available. Detection reagents Millipore s enzymatic detection substrates, Immobilon HRP and Visualizer Spray and Glow, offer you sensitive western blot detection in a variety of formats for ultimate flexibility and quality. 10

11

12 Millipore, Immobilon, Amicon, Catch and Release, Chemicon and Upstate are registered trademarks of Millipore Corporation. SNAP i.d., ReBlot, Spray and Glow, Visualizer, the M mark and Advancing Life Science Together are trademarks of Millipore Corporation. ReNcell is a trademark of ReNeuron Group PLC. Lit. No. PB1017EN00 Rev. A Printed in U.S.A. and France 5/08 LS SBU Millipore Corporation, Billerica, MA. All rights reserved.