Electrophoresis

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<strong>Electrophoresis</strong> · Blotting Membranes Roti ® -PVDF PVDF membrane from ROTH. Roti ® -PVDF is a hydrophobic, microporous polyvinylidene membrane. It is free from adjuvants and detergents. This membrane enables efficient transfer of proteins over a wide molecular weight range. Pore size is 0.45 µm. This mechanically rigid transfer membrane can be easily cut to the desired gel size and it remains flexible and tear-proof even when dry. Membrane thickness 125±25 µm. Briefly immerse Roti ® -PVDF membrane in 100 % methanol prior to transfer until no more white areas are visible. This transfer membrane is then compatible with every aqueous blot system. Applications: Western-blot, Dot-blot, lipopolysaccharide- and amino acid analysis, bindings assays, N-terminal peptide sequencing Detection systems: Radioactive, chemiluminescent, colour Not suitable for use with infrared densitometers (700 nm / 800 nm). Pore size Binding Dimensions L x W Art. No. Pack Qty. € (μm) (cm) 0.45 hydrophobic 375 x 26.5 T830.1 1 roll 276,30 Rapid Immunodetection The following protocol describes a new method of immunodetection, specially designed for PVDF-membranes (Art. No. T830.1 and T831.1). By the use of dry membranes for detection, blocking and some washing steps can be omitted, allowing the procedure to be finished in 2.5 hours. Compatible with chromogenic and chemiluminescent substrates. Following the blotting dry membranes at 37 °C (approx. 1 h) or at room temperature (RT) (approx. 2 h). Membranes must be thoroughly dry. • Incubate in primary antibody (diluted in blocking solution, e.g. 1x Roti ® -Block, Art. No. A151.1) with gentle agitation, 1 h, RT. Be careful to fully cover membranes with antibody solution. • 3 x washing in PBS* for 5 min each, RT • Incubate in secondary antibody (diluted in blocking solution) with gentle agitation, 30 min, RT • 3 x washing in PBS* for 5 min each, RT • Detect immuno-complexes by chromogenic (NBT, Art. No. 4421.1, BCIP Art. No. 6368.1) or chemiluminescent detection (Roti ® -Lumin, Art. No. P078.1) This protocol is not recommended for detection of very low amounts of protein. *PBS: Roti ® -Stock 10x PBS, Art. No. 1058.1 Immobilon-P Original PVDF membrane from Millipore, for outstanding Western Blots. The Immobilon-P membrane consists of microporous polyvinylidene fluoride (PVDF) with a high binding capacity for all biomolecules, particularly for proteins, independently of the transfer method or gel used. The efficient signal-to-noise ratio allows long exposure times and provides a high sensitivity during detection even when working with low protein amounts. Pore size 0.45 µm. Membrane thickness 100-145 µm. Instructions for use: Compared to nitrocellulose the Immobilon-P membrane is much easier to handle and much better to stain, and shows superior resistance to organic solvents. Furthermore, the open pore structure provides easy access of antibodies or other peptides used for detection to bound protein, and enables efficient removal of unbound molecules, efficiently reducing the background. Immobilon-PVDF membrane is ideal when performing immunodetection. The rapid immunodetection method additionally provides a tool to reduce the time span needed for the process by several hours. During Western Blot, the superior qualities of the PVDF membrane not only supersede a blocking step, but also reduce number and time span of washing steps considerably without compromising on specificity or sensitivity. Applications:. Western-blot, Dot- and Slot-blot, amino acid analysis, bindings assays, detection of glycoproteins, N-terminal peptide sequencing. Detection systems: Radioactive, chemiluminescent, colour Pore size Binding Dimensions L x W Art. No. Pack Qty. € (μm) (cm) 0.45 hydrophobic 375 x 26.5 T831.1 1 roll 364,45 Application of ROTH-Transfer Membranes Global Step - Email : Office@global-step.net Telefon : (+4) 0742 095 539 Tel/Fax : (+4) 031 104 85 87 Mobil : (+4) 0720 095 539, 0723 442 593 Biomolecules Nylon plus Nylon 0.2 PVDF Fluoro PVDF NC DNA +++ + - - - RNA + +++ - - - Proteins - + +++ +++ + Detection System Colour +++ +++ +++ +++ +++ Radioactivity +++ +++ +++ +++ +++ Chemoluminescence +++ +++ +++ +++ + Fluorescence +++ +++ - +++ - +++: recommended +: suitable -: not recommended 897 Elektrophorese
Elektrophorese <strong>Electrophoresis</strong> · Blotting Membranes Transfer Membranes made of Nylon For perfect Southern- and Northern-Blots • Very low background • Suitable for all staining techniques • Superior performance in nucleic acid absorption • Available as roll, easy-to-use pre-cut membranes and disks – the perfect size for each application Roti ® -Nylon plus Ideal for DNA/RNA-transfers. Roti ® -Nylon plus is a cationic, nylon-reinforced membrane with 0.45 µm pore size and extremely high tensile strength. The positively charged membrane surface guarantees rapid and efficient binding of DNA and RNA. Also available in convenient pre-cut sheets of standard gel size. Instructions for use: Nucleic acids can be cross-linked to the membrane by baking (80 °C, 15 min) or through UV exposure. No cross-linking is required for alkaline transfer as the negatively charged DNA forms a very stable complex with the positively charged Roti ® -Nylon membrane. Applications: Southern- and Northern-blot, Dot-blot, DNA fingerprinting, macro- and microarrays. Detection systems: Radioactive, chemiluminescent, fluorescent. Pore size (μm) Load Dimensions L x W (cm) Art. No. Pack Qty. € 0.45 positive 300 x 30 K058.1 1 Roll 259,10 0.45 positive 10 x 10 HP34.1 10 Sheets 121,50 0.45 positive 10 x 15 HP35.1 10 Sheets 143,00 0.45 positive 20 x 20 HP36.1 10 Sheets 215,00 0.45 positive 22.2 x 22.2 HP37.1 10 Sheets 246,20 Rotilabo ® -sealing roller Frame made of metal, with rubber roller. For more effective sealing ofmicrotest-, PCR- and Deep-Well-plates with sealing foil or sealing mats as the roller presses them on more evenly. Also usable for evenly smoothing out gel, transfer membrane and blotting-paper during blot-assembly. Width: 10 cm. Art. No. Pack Qty. € HE23.1 1 33,35 898 Global Step - Email : Office@global-step.net Telefon : (+4) 0742 095 539 Tel/Fax : (+4) 031 104 85 87 Mobil : (+4) 0720 095 539, 0723 442 593 Roti ® -Nylon 0.2 Ideal for DNA/RNA transfer, particularly for colony- and plaque-lifting, as well as for all multiple rehybridisations. Especially recommended for Northern-blots. Roti ® -Nylon 0.2 is a neutral, enhanced Nylon membrane. Based on the material it provides a superior tensile strength and is extremely resistant against alcohol, acetone, and many other organic solvents. In a buffer pH of 6.5 Roti ® -Nylon 0.2 is uncharged and by means of the surfacestanding amino- and carboxy-groups has superior hydrophilic qualities. The charge of the membrane (ζ-potential) may be altered through the pH of the buffer - the membrane is positively charged at pH6.5. Membrane of particulary high DNA-binding capacity. Available as roll (pore size 0.2 µm) for high-flexible usage, and as disks (pore size 0.45 µm) for colony- and plaque-lifting. Instructions for use: Covalent bondage of transferred nucleic acids is achieved by cross linking (baking for 15 min at 80 °C or UV-cross linking for 20 sec at 260 nm). Due to the excellent signal-to-noise ratio of the membrane, very sensitive detection (single copy-gene detection) and multiple rehybridisation is possible without loosing sensitivity. Applications: Southern- and Northern blot, Dot blot, DNA fingerprinting, ELISA, colony- and plaque lifting, macro- and microarrays. Detection systems: Radioactive, chemiluminescent, fluorescent. Pore size (μm) Load Dimensions L x W (cm) Art. No. Pack Qty. € 0.2 neutral 300 x 30 AE50.1 1 Roll 269,85 0.45 neutral ∅ 8.2 HP38.1 50 Disks 192,45 0.45 neutral ∅ 13.2 HP39.1 50 Disks 325,75 Size Comparison of Membrane Pores and Biomolecules Biomolecules / Membrane Pores Size (kD) genomic DNA (human) >11000000 E. coli approx. 1500000 0.45 μm approx. 5000 0.2 μm approx. 2000 DNA 3 kb approx. 1000 RNA 1 kb approx. 300 Antibodies (IgG) approx. 150 Proteins in the lab approx. 10 - 300 BSA (Albumin) 69
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Electrophoresis

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