More choices, better detection.

Transcription

1 3 Protein Detection More choices, better detection. Perform Western blotting experiments faster and easier with our new Thermo Scientific Pierce G2 Fast Blotter and accessories, and push your microscopy and cellular imaging to better results with Thermo Scientific DyLight PEGylated Dyes and Conjugates. 16 hermoscientific.com/previewseu Vol. 18, Issue 2014

2 Thermo Scientific and Accessories High performance Western blot protein transfer in 5-10 minutes. The Thermo Scientific Pierce G2 Fast Blotter is designed specifically for rapid semi-dry transfer of kDa proteins from polyacrylamide gels to nitrocellulose or PVDF membranes. The new Pierce G2 Fast Blotter features an integrated power supply optimized to deliver consistent, high-efficiency protein transfer. The system has been verified to work with commonly used pre-cast and homemade SDS-PAGE gels and can be used to simultaneously transfer up to four mini-sized gels or two midi-sized gels. The has an easy-to-use color LCD touchscreen with pre-programmed transfer methods based on the gel number, gel size and molecular weight range of proteins. The easy-touch programming also allows users to quickly create, run and save custom transfer methods. Fast Transfer proteins in 5-10 minutes High transfer efficiency Transfer of low, medium and high MW (10-300kDa) proteins equivalent or better than conventional semi-dry or wet (tank) transfer methods Integrated power supply Provides consistent high efficiency protein transfer Easy-touch programming Access pre-programmed transfer methods based on the gel number, gel size and molecular weight range of proteins using color LCD menu touchscreen. Create, run and save custom transfer methods Convenient Simultaneously transfer up to 4 mini-sized gels or 2 midi-sized gels Versatile Use Pierce 1-Step Transfer Buffer for fast blotting programs or Towbin transfer buffer for conventional semi-dry transfer methods When used in conjunction with the Thermo Scientific Pierce 1-Step Transfer Buffer, the is designed to provide transfer efficiency in 5-10 minutes, which is equivalent to or better than traditional Western blotting techniques. It also eliminates the need for gel pre-equilibration. This significant reduction in protein transfer time is accomplished by optimizing the ionic strength of the transfer buffer and increasing the current [amps (A)/cm 2 ] flowing through the transfer stack. The can also be used for standard semi-dry transfer protocols using Towbin 1 buffer, although transfer times will be longer. Reference 1. Towbin, H., et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76(9): Molecular weight range Low (<25kDa) Mid (25-150kDa) High (>150kDa) High (>150kDa) Gel type Pierce Precise Protein Gel (Tris-glycine) NuPage 4-12% Bis-Tris Gel Criterion 4-20% Tris-HCl Gel NuPage 4-12% Bis-Tris Gel Membrane Nitrocellulose Nitrocellulose PVDF PVDF Target protein and size Cyclophilin B (21kDa) PLK-1 (67kDa) Ecm29 (205kDa) mtor (289kDa) (10 minutes) Trans-Blot Turbo (10 minutes) iblot Transfer (7 minutes) Conventional Semi-Dry (1 hour) Conventional Tank (Overnight) The Thermo Scientific allows rapid transfers of low, medium, and high molecular weight proteins. HeLa lysate was serially diluted, prepared for SDS-PAGE, and electrophoresed according to the gel suppliers recommendations. The proteins were then transferred from gel to nitrocellulose or PVDF membrane using the following techniques: Trans-Blot Turbo Transfer Device with appropriate Trans-Blot Turbo Transfer Pack for 10 minutes; with Pierce 1-Step Transfer Buffer, filter paper and membrane for 10 minutes; iblot transfer with appropriate transfer stack for 7 minutes; classical semi-dry using Towbin transfer buffer (25 mm Tris, 139 mm glycine, 20% methanol), appropriate filter paper and membrane for 1 hour (25V constant); or classical overnight tank transfer using Towbin transfer buffer (30V constant, 16 hours). Membranes were probed with Thermo Scientific anti-cyclophilin B (Product # PA1-027A), anti-plk-1 (Product # MA1-848), anti-ecm29 (Product # PA3-035) and anti-mtor Antibody (Product # PA1-518). The resulting blots were simultaneously imaged using the Thermo Scientific MYECL Imager and densitometry was determined using Thermo Scientific MYImageAnalysis Software. For more detailed technical information on these new products visit thermoscientific.com/pierce 17

3 3 Protein Detection Thermo Scientific and Accessories (cont.) Densitometry (Intensity/Area) 1,200 1, Overnight Tank (16 hours) (10 minutes) Lysate 5µL 1 Lysate µL 10µL 25µL 40µL Anti-HA Magnetic Bead Volume 10µL 25µL 40µL Thermo Scientific provides efficient protein transfer in 10 minutes. GST-PI3K-SH2-HA (37kDa) was expressed in E. coli and purified with varying volumes of Thermo Scientific Anti-HA Magnetic Beads (Product # 88836). Resulting samples were prepared for SDS-PAGE and electrophoresed. The proteins were then transferred from gel to nitrocellulose membrane using either classical overnight tank transfer with Towbin transfer buffer (25mM Tris, 139 mm glycine, 20% methanol) or Pierce 1-Step Transfer Buffer and the (10 minutes). Membranes were probed with anti-ha antibody for 1 hour, washed 5 times, probed with goat anti-mouse HRP for 30 minutes, washed 5 times and incubated in Thermo Scientific SuperSignal West Femto Substrate (Product # 34096). The resulting blots were simultaneously imaged using the MYECL Imager and densitometry determined using MYImageAnalysis Software. Tank Transfer (16 hr.) (10 min.) Sufficient for semi-dry transfer of proteins from polyacrylamide gels to membranes Kit Contents: Blotter and Accessories (Product # 62288): Control Unit Cassette Western Blot Roller Power Cord with C/13 Connector Quick Start Guide Bundled Promotional Items (expires June 30, 2014): Pierce 1-Step Transfer Buffer, 1L, Product # Filter Paper, 7x8.4cm, 100 sheets, Product # part unit Western Blot Roller Sufficient for replacement of G2 Fast Blotter accessory Power Cord with C/13 Connector, Continental Europe Sufficient for replacement of G2 Fast Blotter accessory Power Cord with C/13 Connector, United Kingdom Sufficient for replacement of G2 Fast Blotter accessory 1 roller 1 cord 1 cord Control Unit Sufficient for semi-dry transfer when used with Cassette Kit Contents: Control Unit Western Blot Roller Power Cord with C/13 Connector Quick Start Guide Cassette Sufficient for semi-dry transfer when used with Control Unit 1 unit 1 unit 18 thermoscientific.com/previewseu Vol. 18, Issue 2014

4 Thermo Scientific Pierce 1-Step Transfer Buffer High ionic strength buffer and optimized filter paper for rapid, high-efficiency semi-dry transfer. The Thermo Scientific Pierce 1-Step Transfer Buffer is designed for rapid semi-dry transfer of kDa proteins from polyacrylamide gels (SDS-PAGE) to nitrocellulose or PVDF membranes using the. When used in conjunction with the Pierce G2 Fast Blotter, the transfer buffer provides 5- to 10-minute transfer efficiency, which is equivalent or better than traditional Western blotting techniques. The Pierce 1-Step Transfer Buffer is also compatible with other semi-dry transfer devices with a suitable high-current power supply for fast protein transfer. Efficient High ionic strength buffer specifically designed for rapid semi-dry protein transfer Versatile Optimized for 5-10 minute protein transfer using the Pierce G2 Fast Blotter, compatible with other semi-dry transfer devices and a suitable high-current power supply for fast protein transfer Economical High performance without special or costly consumables required by other fast semi-dry transfer devices High-current power supply: 1.3amps (A) required to transfer maximum surface area of 60cm 2 (one mini-sized gel); 2.5A required to transfer maximum surface area of 120cm 2 (two mini-sized gels or one midi-sized gel); 3.8A required to transfer maximum surface area of 180cm 2 (three mini-sized gels); 5.0A required to transfer maximum surface area of 240cm 2 (four mini-sized gels or two midi-sized gels) Pierce 1-Step Transfer Buffer Sufficient for 20 mini blots at 50mL each Pierce 1-Step Transfer Buffer Sufficient for 4 mini blots at 50mL each 1L 200mL Thermo Scientific Pierce Western Blotting Filter Paper The Thermo Scientific Pierce Western Blotting Filter Papers are pre-cut cotton sheets for wet or semi-dry, passive or electrophoretic transfer of proteins from polyacrylamide gels (SDS-PAGE) to PVDF, nitrocellulose or other membranes. High-quality Pierce Western Blotting Filter Papers are available in two convenient sizes for use with mini-sized or midi-sized gels. The filter paper is recommended for use with the, which is designed to provide highly consistent protein transfer using transfer stacks that do not exceed 1.8mm (two sheets of ~0.83mm filter paper) total filter paper thickness for the bottom of the stack or 1.8mm (two sheets of ~0.83mm filter paper) total filter paper thickness for the top of the stack. Validated performance Tested and recommended for use with the to provide extremely consistent protein transfer Pre-cut Available in two convenient sizes for use with mini-sized or midi-sized gels High-quality Clean cotton cellulose fiber paper manufactured with additive-free ultrapure water to eliminate sources of background signal and artifacts Western Blotting Filter Paper, 7cm x 8.4cm Sufficient for optimal transfer performance with the Western Blotting Filter Paper, 8cm x 13.5cm Sufficient for optimal transfer performance with the 100 sheets 100 sheets For more detailed technical information on these new products visit thermoscientific.com/pierce 19

5 3 Protein Detection Thermo Scientific DyLight PEGylated Reactive Dyes and Conjugates Fluorescent dyes for applications requiring biocompatibility (i.e., nontoxic), enhanced fluorescence, low quenching and reduced nonspecific binding. Thermo Scientific DyLight PEGylated Dyes are derivatives of the high-performance Thermo Scientific DyLight Dyes that can be used to fluorescently label antibodies and other proteins. Conjugates made with DyLight PEGylated Dyes can be used as molecular probes for cellular imaging and other fluorescence detection methods. DyLight PEGylated Dyes contain two to four nontoxic polyethylene glycol (PEG) chains. The PEG chains enhance fluorescence, reduce nonspecific binding of conjugates, improve solubility of the dyes and labeled molecules in aqueous solution, aid in cell permeability, and improve retention, especially in tumors. 1 The near-infrared (NIR) to far-red fluorescence properties of some of the PEGylated dyes such as Thermo Scientific DyLight 680, 755 and 800 Dyes make them useful in biological, chemical and pharmaceutical applications, including in vivo imaging. The DyLight PEGylated Amine- and Sulfhydryl-Reactive Dyes are excellent alternatives to Alexa Fluor, IRDye and CF Dyes, providing superior fluorescence characteristics (e.g., brightness, photostability, ph insensitivity and water solubility). In addition, the high-fluorescence intensity of DyLight PEGylated Dyes provides outstanding sensitivity and requires less conjugate in most applications. Soluble PEGylated dyes and labeled molecules improve solubility in aqueous solution, aid in cell permeability and improve retention, especially in tumors High fluorescence intensity Significantly brighter fluorescence than LI-COR IRDye and Alexa Fluor Dyes Efficient labeling methods Well-characterized chemistry and optimized protocols provide for reliable, high-quality labeling Reference 1. Knop, K., et al. (2010). Poly(ethylene glycol) in drug delivery: Pros and cons as well as potential alternatives. Angew Chem Int Ed 49: Properties of the Thermo Scientific DyLight PEGylated NHS-Ester Fluors DyLight Fluor Ex/Em* ε MW (g/mol) Spectrally Similar Dyes DyLight 550-2xPEG Dye 557 / , Alexa Fluor 555, Cy 3, DyLight 550, CF555 Dyes DyLight 650-4xPEG Dye 658 / , Alexa Fluor 647, Cy5, DyLight 650, CF647 Dyes DyLight 680-4xPEG Dye 684 / , Alexa Fluor 680, Cy5.5, DyLight 680, CF680, IRDye 680 Dyes DyLight 755-4xPEG Dye 754 / , Alexa Fluor 750, DyLight 755, CF750 Dyes DyLight 800-4xPEG Dye 783 / , Alexa Fluor 790, Cy7, DyLight 800, CF790, IR Dye 800 Dyes * Excitation and emission maxima in nanometers. Molar extinction coefficient (M -1 cm -1 ) Properties of the Thermo Scientific DyLight PEGylated Maleimide Fluors DyLight Fluor Ex/Em* ε MW (g/mol) Spectrally Similar Dyes DyLight 550-2xPEG Dye 557 / , Alexa Fluor 555, Cy3, DyLight 550, CF555 Dyes DyLight 650-4xPEG Dye 656 / , Alexa Fluor 647, Cy5, DyLight 650, CF647 Dyes DyLight 680-4xPEG Dye 684 / , Alexa Fluor 680, Cy5.5, DyLight 680, CF680, IR Dye 680 Dyes DyLight 755-4xPEG Dye 757 / , Alexa Fluor 750, DyLight 755, CF750 Dyes DyLight 800-4xPEG Dye 784 / , Alexa Fluor 790, Cy7, DyLight 800, CF790, IR Dye 800 Dyes * Excitation and emission maxima in nanometers. Molar extinction coefficient (M -1 cm -1 ) 20 thermoscientific.com/previewseu Vol. 18, Issue 2014

6 DyLight 650-4xPEG DyLight Fluorescence Intensity Signal/Background CF 647 Alexa Fluor 647 S/B DyLight 650-4xPEG DyLight 650 CF-647 Alexa Fluor X X X X Thermo Scientific DyLight 650-4xPEG Dye is brighter than other spectrally similar fluors. The endoplasmic reticulum of U20S osteosarcoma cells was fluorescently labeled with mouse anti-pdi primary antibody and goat anti-mouse secondary antibody conjugated to the indicated dyes. All images were captured using the same exposure time and contrast and brightness settings. Thermo Scientific DyLight 650-4xPEG-GAR shows a much lower rate quenching. The conjugates show excellent staining specificity and higher fluorescence intensity at high dye molar excesses. At optimal molar excess DyLight 650-4xPEG GAR signal/background is about three-fold higher than CF 647-GAR and Alexa Fluor 647-GAR conjugates DyLight 550-2xPEG NHS Ester DyLight 550-2xPEG Maleimide DyLight 650-4xPEG NHS Ester Sufficient for one use to label a total of 16mg of IgG DyLight 650-4xPEG Maleimide Sufficient for one use to label a total of 16mg of IgG DyLight 755-4xPEG NHS Ester DyLight 755-4xPEG Maleimide DyLight 800-4xPEG NHS Ester Sufficient for one use to label a total of 12mg of IgG DyLight 800-4xPEG Maleimide Sufficient for one use to label a total of 12mg of IgG SA SA Goat anti-mouse IgG (H+L) Secondary Antibody, DyLight 800-4xPEG conjugate Goat anti-rabbit IgG (H+L) Secondary Antibody, DyLight 800-4xPEG conjugate 1mL 1mL Note: Product # will be replaced by SA and will be replaced by SA The replaced products have been updated with the DyLight 800-4xPegylated Dye for improved antibody stability and brightness. For more detailed technical information on these new products visit thermoscientific.com/pierce 21