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Western Blotting ptglab.com 1 The Complete Guide To WESTERN BLOTTING www.ptglab.com 2 Western Blotting ptglab.com 3 WELCOME About this guide This booklet aims to give you a complete guide on how to perform a Western blot that produces optimal results. The booklet includes original Western blot lab protocols, useful technical tips, and troubleshooting. This guide will take you through the experiment step by step, enabling you to get the most from your research. What’s Inside 6–9 Standard Western Blot Protocol 10–11 How To Optimize Your Western Blot 12–13 SDS-PAGE Gel Recipes 14–15 How To Optimize Your Results With Low MW Proteins 16 Tricine Gel Recipe For Low MW Proteins 17 Choosing The Right Lysis Buffer 18–23 Troubleshooting 26–30 Loading Control Antibodies 31 Contact Us 4 Western Blotting THE BENCHMARK IN ANTIBODIES Since the day it was founded, Proteintech®* has been making all of its products to the highest standards possible whilst taking complete responsibility for the quality of each product. • Proteintech makes every single antibody in its 12,000+ catalog. • Each Proteintech product is unique and cannot be bought under a different label. • Antibodies are detected with siRNA-treated samples to demonstrate specificity. • It works in every single species and application or get a full money-back refund. Proteintech has over 12,000 antibodies in its extensive catalog, all fully validated and available for next day delivery. www.ptglab.com *Proteintech® and the Proteintech logo are trademarks of Proteintech Group registered in the US Patent and Trademark Office. ptglab.com 5 6 Western Blotting Standard Western Blot PROTOCOL SDS-PAGE 1. Construct an SDS-PAGE gel according to the molecular weight (MW) of your target protein(s). See page 12 for SDS-PAGE gel recipes. 2. Prepare samples in microfuge tubes. Add 4x SDS sample buffer so the total protein amount is 30–50μg per sample (according to the protein amount measured by Bradford or BCA protein assay). 3. Flick microfuge tubes to mix samples, spin them shortly, and then heat to 95–100°C for 5 minutes. 4. Set up electrophoresis apparatus and immerse in 1x running buffer. Remove gel combs and cleanse wells of any residual stacking gel by pipetting running buffer up and down in each well using gel-loading tip (Figure 1). 5. Load samples and appropriate protein markers onto the gel using a tip. 6. Place the lid on the gel tank. Turn on electrophoresis power pack and set to a low voltage (as the sample runs through the stacking gel), increasing to a higher voltage (e.g., 120V) when the dye front reaches the separating layer. Stop the gel running when the dye front migrates to the desired position. Tip: Tris-tricine gels separate low MW proteins (<20 kDa) better than Tris-glycine gels. See page 16. Membrane Transfer Please note: PVDF membranes (or PSQ membranes with 0.22μm micropores for targets less than <30 kDa) are strongly recommended. 1. Soak membranes in methanol for 30 seconds before moving to transfer buffer. 2. Soak the filter papers and sponges in transfer buffer. 3. Sequentially assemble the transfer constituents according to the illustration shown on the next page (Figure 2), and ensure no bubbles lie between any of the layers. Apply semi-dry or wet transfer systems according to the instructions of the blotting apparatus manufacturer. ptglab.com 7 Figure 1 After removing gel combs rinse each well thoroughly with running buffer to ensure no remaining bits of acrylamide are blocking them. Use gel-loading pipette tips to facilitate easy and even loading of your protein samples. Figure 2 A representation of the components of a transfer “sandwich.” Note the orientation of the gel and membrane: the PVDF membrane is situated nearest to the positive electrode. The binding of SDS to proteins results in the complex having an overall negative charge. Therefore, the SDS- bound proteins travel towards the positive electrode. 8 Western Blotting Immunoblotting 1. Following transfer, wash the membrane twice with distilled water. 2. Gently mark the bands of the MW ladder on the membrane using a pencil. 3. If desired, stain the membrane with commercial Ponceau red solution for 30 seconds to visualize protein bands, then wash away Ponceau red solution with generous amounts of 1x TBST. 4. Block with 1x TBST containing (2–5%) non-fat dry milk (or 1–5% BSA for the detection of phospho-epitopes) with constant rocking for 1 hour or overnight at 4°C. 5. Dilute primary antibody in blocking solution with a starting dilution ratio of 1:1000. (Optimal dilutions should be determined experimentally with a dilution series.) Incubate the membrane with primary antibody for 1 hour (or overnight at 4°C) on a benchtop rocker. 6. Wash membrane three times with 1x TBST for 10 minutes each. 7. Incubate the membrane with a suitable HRP-conjugated secondary antibody (recognizing the host species of the primary antibody), diluted according to the instructions. Incubate for 1 hour with constant rocking. 8. Wash membrane three times with 1x TBST for 10 minutes each. Tip: Do not let the membrane dry at any stage of the blotting process. Signal Detection 1. Prepare ECL substrate according to the manufacturer’s instructions. 2. Incubate the membrane completely with substrate for 1–5 minutes (adjust time for more sensitive ECL substrates, e.g., SuperSignal West Femto Chemiluminescent Substrate [Pierce]). 3. Expose the membrane to autoradiography film in a darkroom or read using a chemiluminescence imaging system. 4. Line up the developed film in the correct orientation to the blot and mark the bands of the MW ladder directly onto the film. It is also advisable to add notes such as lane content, film exposure time, and ECL properties. Tip: Use multiple exposure lengths to determine the optimal exposure time. Use fluorescent markers and clip the top- right corner of your film as a guide for blot film orientation. ptglab.com 9 Standard Western Blot PROTOCOL Buffers Required 1x TBST (1000ml) 20mM Tris-base 2.4 g 150mM NaCl 8.76 g 50mM KCI 3.73 g 0.2% Tween-20 2ml Adjust pH to 7.6. Add ddH2O to 1000ml 4x SDS Sample Buffer (100ml) 150mM Tris HCl (pH 7.0) 15ml (of a 1M stock) 25% Glycerol 25ml 12% SDS 12 g 0.05% Bromophenol Blue 0.05 g 6% ß-mercaptoethanol 6ml Add ddH2O to 100ml, aliquot, and store at -20ºC. Wet Transfer Buffer (1000ml) 25mM Tris-base 3.03 g 192mM Glycine 14.4 g 20% Methanol 200ml Add ddH2O to 1000ml. Semi-dry Transfer Buffer (1000ml) 48mM Tris-base 5.81 g 39mM Glycine 2.93 g 0.0375% SDS 0.375 g 20% Methanol 200ml Add ddH2O to 1000ml 10 Western Blotting How To Optimize Your WESTERN BLOT Obtaining perfect, publication-ready results straightaway is not exactly the norm when performing Western blotting. However, there are steps you can take to refine your blotting experiments and ensure they get off to the best start possible. 1. SDS-PAGE Your Western blotting experiments start long before you begin to work with your membrane. If your gel is sloppily made or you run it at an excessive voltage, your final results will suffer. If you cast your own gels, take extra care to ensure uniformity in their makeup. Select the right acrylamide percentage and avoid that dye-front “smiling” effect by resisting the temptation to run your gels at high voltages. Above all, load a uniform amount of protein; the Proteintech® validation lab finds that 0–50ug of protein is a suitable amount for the detection of most proteins, and usually gives streak-free and well-separated bands. 2. Membrane Membrane choice can make a huge difference to the outcome of your Western blotting experiments. The two main membrane types are PVDF and nitrocellulose, but there are now several versions of each on the consumables market. PVDF tends to be good for lowly expressed proteins, but high background and it works better with hydrophilic/polar/charged target antigens. Nitrocellulose membranes are good for normal or highly expressed proteins and it works better with hydrophobic/ non-polar antigens. 3. Antibody Concentration Your next step should be to determine the optimum working antibody concentration. The rate of binding between antibody and antigen is affected by their relative concentrations in solution (among other variables). Often you will need to tweak the antibody concentration to get better blots. This step is called antibody titration, and it should be performed every time a new antibody or new set of experimental conditions is used. “Often you will need to tweak the antibody concentration to get better blots.” ptglab.com 11 “Remember that milk-based blocking buffers contain such things as endogenous biotin, glycoproteins, and enzymes that may interfere with the signal.” 4. Blocking Conditions Various blocking buffers are available and not all of them work in every situation. It is important to try several blocking buffers for each antibody-antigen pair. Remember that milk- based blocking buffers contain such things as endogenous biotin, glycoproteins, and enzymes that may interfere with the signal. Milk-based buffers, for example, contain active phosphatases that will sabotage your detection of phosphorylated proteins; in these cases, try alternatives such as bovine serum albumin- based blockers. 5. Washing If the Western blot shows high background signal, revising the washing steps on the next run-through has proven to be useful. Increasing washing times and volumes will reduce background signal along with performing additional buffer changes or using a stronger detergent (e.g., SDS instead of Tween 20). 6. Detection The detection stage is integral to obtaining a good Western blot signal.
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