western blotting Protein transfer technical handbook separate transfer detect Pre-transfer Transfer systems Post-transfer Protein transfer technical handbook 3 Comprehensive protein transfer solutions Contents designed to drive your success Introduction to electrotransfer methods for Western blotting 4 Protein transfer is a vital step in Western blotting Pre-transfer considerations 6 involving the transfer of proteins separated Choice of electrotransfer system 6 in a gel by electrophoresis to a solid support Building the transfer sandwich 7 Choosing the Western blot membrane 7 matrix so that specific proteins can be detected Nitrocellulose membranes 8 using immunodetection techniques. We offer PVDF membranes 10 Blotting paper 10 a complete array of products to support rapid Protein ladder considerations 12 and efficient protein transfer for Western Transfer buffers 14 blotting. Our portfolio of high quality protein Transfer systems 16 transfer products unites membranes, buffers, Wet electroblotting 18 Semi-dry electroblotting (semi-dry transfer) 21 stains, molecular weight markers, alongside Dry electroblotting (dry transfer) 24 a comprehensive choice of transfer devices Post-transfer 27 designed to suit your needs and enable better Monitoring transfer efficiency 27 Signal enhancements 30 quality Western blot results. Appendix 36 Protocol quick reference 36 Troubleshooting 40 Ordering information 42 For a complete listing of all available products and more, visit thermofisher.com/western 4 Pre-transfer Transfer systems Post-transfer Protein transfer technical handbook 5 Introduction to electrotransfer methods for Western blotting Protein transfer from gel to membrane is necessary for two The transfer of size-separated Prepare transfer buffer reasons: 1) better handling capability offered by the membrane than the fragile gel during Western blot processing and 2) better Prepare transfer buffer sufficient for the proteins from a polyacrylamide transfer tank and for equilibration of gels target accessibility on the membrane by macromolecules like and membranes. gel to a membrane support is one antibodies. In general, all electrotransfer methods rely on the electrophoretic mobility of proteins to move them out of a gel. The of the key steps to subsequent techniques involve placing a protein-containing polyacrylamide Equilibrate gels and membranes gel in direct contact with a piece of nitrocellulose membrane, Equilibrate gels and membranes immunodetection of a specific polyvinylidene difluoride (PVDF) membrane or other suitable in transfer buffer. protein-binding support. Next, the gel-membrane element is Support Grid “sandwiched” between two electrodes, which are typically Pads protein via Western blotting. Assemble the gel and membrane sandwich Filter Paper submerged in a conducting solution (transfer buffer) (Figure 1). Transfer Membrane Gel Scientists have used a variety When an electric field is applied, the proteins move out of the Place the membrane and gel between gel and onto the surface of the membrane, where the proteins buffer-soaked filter papers. of methods for this transfer become tightly attached. The resulting membrane is a copy of the protein pattern that was in the polyacrylamide gel. For a complete Set up the transfer cell workflow, see Figure 2. step, including diffusion transfer, Gel/Membrane/Filter Paper Place the gel, membrane and filter paper Sandwich Gel sandwich in the transfer tank. Fill the tank capillary transfer, heat-accelerated Transfer Membrane with transfer buffer. Connect the Filter Paper tank to the power supply and set the power Pads supply for optimal power and time. Buffer Tank convectional transfer, vacuum Support Grid Figure 1. Western Anode (+) Gel/Membrane/Filter Did you know? Electrodes Sandwich blot electrotransfer of W. Neal Burnette in blotting and electroblotting Start the transfer Cathode (-) proteins from gel to Tobert Nowinski’s lab at the membrane. While this Direction of Transfer Fred Hutchinson Cancer (electrotransfer). Among these diagram depicts the Buffer Tank Figure 2. Workflow of the tank electrotransfer of proteins for Research Center in Seattle was setup of a typical wet Western blotting. transfer, many of the the first to coin the term methods, electroblotting has Anode (+) “Western blotting.” The term Electrodes principles apply to semi- Cathode (-) dry and dry methods There are three ways to electrotransfer proteins from SDS-PAGE or honors Edwin Southern, who emerged as the most popular of protein transfer to native gels to nitrocellulose or PVDF membranes for the purpose described blotting of DNA, and Direction ofr Transfe membranes. of Western blotting: is a reference to the West Coast because it is faster and more location of Nowinski’s lab. • Wet electroblotting (traditional wet or tank transfer) efficient than the other methods. • Semi-dry electroblotting (semi-dry transfer) • Dry electroblotting (dry transfer) This handbook will focus on these three techniques and other considerations to help improve your protein transfer efficiency for better Western blot results. Learn more at thermofisher.com/western 6 Pre-transfer Transfer systems Post-transfer Protein transfer technical handbook 7 Pre-transfer considerations Table 1. Characteristics of our electrotransfer systems. In general, dry and semi-dry transfer methods are Choice of Traditional wet transfer Rapid semi-dry transfer Dry transfer faster than wet techniques. If time is a factor, you may electrotransfer system want to consider these methods. However, the speed of transfer may affect transfer efficiencies. It is typically perceived that the slower, wet transfer methods tend When setting up a Western to offer better transfer efficiencies than the faster semi-dry or dry transfer methods. If the amount of blot experiment, one of the target or antibody quality is an issue, you may want to first decisions is the choice of choose a wet transfer method or consider other signal enhancements (see page 30). electrotransfer method: wet, XCell II Blot Module Mini Blot Module Pierce Power Blotter iBlot 2 Gel At Thermo Fisher Scientific, we offer transfer systems Transfer Device semi-dry or dry. This choice for the three electrotransfer methods. See Table 1 for a comparison of our systems and read more about the Transfer 60–120 min 60 min 5–10 min 7 min will drive what transfer buffers individual transfer methods beginning on page 16. time (or lack thereof) will be used Power External External Internal Internal supply and may impact the choice of membrane products because Building the Choosing the some transfer devices have Western transfer sandwich Western blot membrane specific requirements. The choice After proteins have been separated by electrophoresis, the next The most common immobilization membranes for Western blotting step is the assembly of the “transfer sandwich.” For wet and are nitrocellulose, polyvinylidene difluoride (PVDF) and nylon. The of transfer device may also drive semi-dry electrotransfer devices, the transfer sandwich typically following characteristics make these membranes suitable for Did you know? consists of a filter paper-gel-membrane-filter paper arrangement, protein transfer: the gel size, blotting area and Harry Towbin’s group in Basel, where the filter paper aids in wicking of the transfer buffer. Switzerland, was the first to For some commercially available wet and semi-dry transfer • A large surface area to volume ratio describe electroblotting of throughput, as well as potentially devices, pre-assembled transfer stacks are available in which the proteins to membranes and the • A high binding capacity polyacrylamide gel is inserted. impact transfer efficiencies. use of secondary antibodies. • Extended storage of immobilized proteins The method he described in Dry transfer devices do not require filter paper for wicking transfer 1979 is the closest technique to • Easy to use buffer. Instead a unique gel matrix transfer stack incorporates what we know as modern-day buffer, eliminating the need for buffer tanks and wetted filter paper. • Possible optimization for low background, signal Western blotting. and reproducibility Western blot membranes are typically supplied in either sheets or rolls, and commonly have a thickness of 100μm, with typical pore sizes of 0.1, 0.2 or 0.45μm. Most proteins can be successfully blotted using a 0.45μm pore size membrane, while a 0.1 or 0.2μm pore size membrane is recommended for low molecular weight proteins or peptides (<20kDa). For some transfer instruments, prepackaged membranes and blotting paper “stacks” are available. Table 2 provides a summary of available membranes. Learn more at thermofisher.com/transfer 8 Pre-transfer Transfer systems Post-transfer Protein transfer technical handbook 9 Table 2. Characteristics of our Western blotting membranes. Nitrocellulose membranes Table 3. Applications and specifications for our nitrocellulose membranes. Nitro- High protein-binding affinity, compatibility with a variety Ready-to-use iBlot 2 Membrane Applications Reprobe Binding Specs Pre-cut nitrocellulose cellulose of detection methods, and binding affinity of proteins and sandwich Transfer Stacks type and uses characteristics interaction roll glycoproteins make nitrocellulose a popular matrix. Protein immobilization on the membrane is thought to occur by LC2009 LC2006 Nitrocellulose Western, Can be Hydrophobic Cat. No. 77012 88013 88024 77010 88014 88025 88018 IB23002 IB23001 hydrophobic interactions. Use of high salt and low methanol LC2000 LC2001 membranes Southern stripped and