The Popular Technology SDS PAGE & Western blotting :Principle and ApplicationCurrent Protocols in Molecular Biology 10.8.1-10.8.28, July 2008 Western Blot Definition: Western blotting is a technique used to identify and locate proteins based on their ability to bind to specific antibodies. 1 Western blot analysis can detect your protein of interest from a mixture of a great number of proteins (a cell can contain 30,000 different proteins - and these same proteins can even be altered giving you over 300,000 different proteins!). an idiom in Chinese (大海撈針) Western blotting can give you information about the size of your protein (with comparison to a size marker or ladder in kDa), and also give you information on protein expression (with comparison to a control such as untreated sample or another cell type or tissue). Summary: Western Blot Gives You Information on the: · Size of your Protein · Expression Amount of your Protein 2 Western blot analysis can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro. Western blot is dependent on the quality of antibody you use to probe for your protein of interest, and how specific it is for this protein. Antibodies are now easily obtainable from commercial sources, and you can purchase one for your protein of interest. Antibodies specific to your protein are vital to western blotting as they are able to bind specifically to your protein of interest instead of the thousands of proteins on your western blot! Figure 1. Details of the steps involved in obtaining protein for western blot. 3 causing the molecules to move toward the positive electrode (the anode.Definition: SDS-PAGE is an abbreviation for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) Electrophoresis is a technique for separating. 3) Electrical current is applied to the gel. SDS-PAGE • Stands for “SDS Polyacrylamide Gel Electrophoresis • Similar in many ways to agarose gel electrophoresis that we used to separate DNA molecules: 1)Thin gel separates molecules on the basis of size. usually marked red) 4 . 2) Negatively charged molecules are loaded into wells in a gel that is submerged in buffer. 5 .Thus.SDS-PAGE 4) Smaller molecules move faster through the gel than larger molecules . smaller molecules will be found at the bottom of the gel and larger molecules will be found at the top of the gel. N'-methylene-bis-acrylamide (bis for short). 6 .In forming the polyacrylamide gel. acrylamide monomers polymerize into long chains that are covalently linked by the crosslinker N. 7 . 8 . Analysis of protein samples by SDS polyacrylamidegel electrophoresis and Western blotting Protein bands detected by specific antibody SDS-PAGE Western blot 9 . To Detect your Protein: · Buy an Antibody Against Your Primary Antibody · Use an ECL .Chemiluminescence Kit and Film to Get the Results 10 . Western blot analysis examines proteins. in order done for western blot: Steps in Western Blotting: -Preparing for Western Blot -Which Antibody to use for Western Blots -Lysing Cells for Western Blot -SDS-PAGE Gel Information -SDS-PAGE Gel Preparation for Western Blots -Gel Electrophoresis . For non-adherent cells you can gently pellet them with centrifugation and then wash the pellet with PBS. and so we want the proteins to be release from the cells and also prevent the proteins from being cut up by proteases. For adherent cells. simple wash the flask or dish with PBS prior to western blot. you need to remove extraneous proteins from the media. 11 .Table of Contents.running the gel -Tranfer of Proteins to Membrane for Western Blotting .Western Blot To Prepare Samples for Western Blot: Whether you have Non-adherent Cells (such as T-cell lines or peripheral blood cells) or Adherent cells (such as Fibroblast cells or COS cells). Buffer -Inhibitors (both protease inhibitors and phosphatase inhibitors if we will do western blot for phospho-proteins) Detergent .have higher avidity (親合力) 12 .overall cleaner results on the western blotting film (less nonspecific bands detected) Polyclonal Antibodies are better suited for Immunoprecipitation: .To Prepare Samples for Western Blot: continue We need these to western blot: . or use an antibody which is conjugated to biotin. Usually either mouse monoclonal antibodies or rabbit polyclonal antibodies are used. You can use either an antibody without any added groups.better signal to noise ratio ( lower background in western blot ) .to keep things cold! 4C on ice during cell lysis for western blotting . Monoclonal Antibodies are usually better for Western Blotting due to: .their higher specificity (you get less contaminating proteins or) . Which Antibody should I use for Western Blotting? You should select an Antibody to use for Western Blot.they recognize more epitopes .Lysing Cells For Western Blot SDS and RIPA are detergents that are used to solubilize proteins for later analysis using western blotting.use Detergent to break up membranes of cells to release proteins . so we need to release these proteins to be able to immunoblot for them. This is required as many proteins are either inside the cell or located inside cell membranes. 1% of SDS or greater can interfere with protein measurement 13 .collect in eppendorf tubes and label (keep these on ice ) Measure total protein before Western Blot Analysis: -this allows more quantitative analysis if you want to compare treatment conditions in western blot analysis -commercial kits are available such as a Bradford assay for measuring total protein (from Bio-Rad) .use an ice bucket -Rule of thumb for how much lysis buffer to use is: 10^5 cells /uL of lysis buffer .0.Lysing: -can be done directly on the plate or dish -pellet the cells -Keep on Ice and cold . This allows proteins to be separated by size and not by charge. Low percentage gels separate larger proteins whereas higher percentage gels separate smaller proteins better. .Poke a hole in cap of each tube to prevent popping SDS-PAGE Gel Information for Western Blotting SDS-PAGE Gels are gel matrices which are used to separate proteins by size in the presence of electric current.Boil in water bath or heating block (lower temperatures such as mid . SDS binds to proteins every few amino acids and neutralizes the charge differences that proteins have. and in an electrical current this could cause problems. This is solved by the addition of SDS to the protein samples. 2% SDS) -add disulfide reducing agent such as beta – mercaptoethanol . The problem is that all proteins have a charged associated with them. 14 .90 degrees decrease protein aggregation ).Denature Protein Samples : -add protein loading sample buffer to an aliquot of cell lysate .contains dye (too see the migration on a gel. Load every Sample carefully and slowly to prevent sample leaking out of the lane.8 is used to pack proteins in together after loading .the percentage of acrylamide is important.SDS-PAGE Gel Preparation for Western Blot .depending on how much protein you will want to load for western blotting.A resolving gel is used at the bottom with a pH of 8. you should use small combs or larger combs. Usually 10% acrylamide is used. .Load every lane and use sample buffer in each (to prevent differences in ). .Polymerization of gels is increased by the catalysts APS and TEMED which speed up the polymerization reaction (formation and solidification) of the poly-acrylamide gel. . . Percent acrylamide (resolving gel) Size range transferred (∼100% efficiency) 29–150 kD 14–66 kD <36 kD <20 kD 5–7 % 8–10 % 13–15 % 18–20 % 15 .8 -A stacking gel (4-5%) pH 6. Watch protein marker/ladder or dye front for when to stop gel. leading to poor resoloving and blurry badly formed bands) 16 . Constant current gives better and sharper results if you have the time.if not you have switched the leads and can lose all your samples! .Gel Electrophoresis Centrifuge samples for 10 sec after boiling. this gives sharper bands. Load sample into each lane Load MW marker Run gel at 100V (constant voltage) or even better at 40 mA (constant current). .Initially run slowly through the stacking gel (~ 50 Volts).Do not run overnight .Do not over-heat the gel (this causes the gel to lose rigidity. Gel Electrophoresis -watch for bubbles between glass and under the gel this means that it is working -watch for protein migration (should be going down) . How an SDS-PAGE gel is run 17 . Tranfer of Proteins to Membrane for Western Blotting Select Membrane Type: Can use either: -PVDF membrane for western blots (hydrophobic) -Nitrocellulose membrane for western blots -Nylon membrane (rarely used now .can tear) Tips for Transfering to Western Membrane: -Wear Gloves at all times! Use forceps .Minimize touching/forceps to the membrane 18 . Assemble the immunoblot sandwich (-) sponge on black filter paper Gel filter paper sponge (+) ? PVDF membrane 19 . 20 . Lyse the cells to release protein contents. Primary antibody Wash Secondary antibody Wash detection 21 . What You Need to Western Blot: · A Protein Sample · A Good Antibody to Detect your Protein of Interest If your protein is a novel protein. Then transfer these gel proteins onto a membrane using electricity. In this case you will need at least a small amount of your protein either purified from cell extracts or made as a recombinant (ie in vitro or in a recombinant protein expression system).How Does Western Blotting Work? See Diagram 1 below. Obtain a protein sample you want to analyze. such as cell samples. Run these on a gel which separates proteins on the basis of size. This membrane can then be used to probe for proteins of interest using a primary antibody. you must produce an antibody yourself or get a company to do it for you. Diagram 1 Membrane (carrier) Diagram 2 ? 22 . As you can see the protein in lane 3 has a higher expression than the normal sample in lane 5. which is interesting. Our protein of interest is also 80 kDa. Also. the protein spots in lanes 3 and 5 are the same size as the 2nd spot in the size ladder from lane 1. So we know that the western blot worked and that the protein is highly expressed in a cancer sample! ? 23 . Lane 1 is a protein size marker ladder which shows different known sizes of proteins. this can be purchased commercially and the sizes of all the spots are given.Diagram 2 shows a western blot example gel. Lane 3 is a cancer sample and lane 5 is a normal sample. For these membranes. This wetting procedure works for nitrocellulose and nylon membranes only. Assaying for Results -usually ECL (Enhanced Chemiluminescence) is used . Do not let membrane dry out at any time. Phospho-proteins can require 2 . 24 .20 minute exposures. PVDF membranes are hydrophobic and will not wet simply from being placed into distilled water or transfer buffer.film is exposed and developed. air will be trapped and will appear as white blotches in the membrane. then equilibrate 10 to 15 min with transfer buffer. If this occurs. Exposure for 10 seconds is usually enough for total protein. Protein will not transfer onto these areas. Place into distilled water slowly. first immerse 1 to 2 sec in 100% methanol.If the membrane is placed into the water too quickly. with one edge at a 45◦ ◦ angle. wet once again with methanol and transfer buffer as described above. substrate 25 .chromogenic Chromogenic. Chromogenic detection 26 . 27 . 28 . Demonstrate enrichment of well-characterized antibody by sampling..3 Applications of Immunoblotting Application Comment Antibody development and characterization Characterize antibody specificity Subcellular localization of an expressed protein Analyze isolated organelles (e.Table 10. electrophoresing. and blotting at various stages of purification Separate and blot a viral lysate to test serum for antibodies that bind key viral proteins.g.g. plasma membrane.8. indicating presence of antibodies against an infection (e.. nucleus) for presence of specific proteins. Golgi apparatus. HIV testing) Protein purification Diagnostics 29 . probe intact cells and tissue for subcellular localization. . peptide sequencing by MALDI (Carr and Annan. track markers and reporter genes (e..g. 1996) Troubleshooting Western Blot Spots on Film (missing bands) : -Poor Transfer due to air bubbles during transfer to membrane -dirty film when adding to the developer .Application Gene expression Comment Detect presence or absence and amount of a specific protein during gene expression.developer rolls are dirty 30 . e.g. luciferase) in transgenic organisms to get a complete picture of transcription and translation Phosphoproteomics: determine phosphorylation status of the protein complement in the cell or tissue using antibodies specific for phospho amino acids Post-translational Modifications Protein sequencing by mass spectrometry Characterize the readily available proteins blotted onto membranes using sensitive methods. The blot should have been blocked with 5% nonfat dry milk prior to treatment. Chromogenic development leaves a permanent stain on the membrane that is difficult to remove. and should not be used when reprobing. Bands Smeared. All residual antibodies are removed from the membrane by first rewetting it in water and then briefly treating it with NaOH. up to five reprobings are generally feasible. 31 . Bands not Sharp -bands smeared due to hot gel .Fuzzy Bands. The stain can interfere with subsequent analysis if reactive bands from sequential immunostainings are close together.bands fuzzy due to high voltage STRIPPING AND REUSING MEMBRANES Reprobing PVDF membranes that have been developed with chemiluminescent reagents is simple and straightforward. Although repeated reprobing can lead to loss of signal. 2 M NaOH 1. Transfer to 0. 2. In order to effectively reprobe the membranes.Materials 0. Wash blot 5 min in distilled water. casein (for AP systems) or nonfat dry milk must be used as the blocking agent. 4.2 M NaOH and wash 5 min. 32 . 3. Wash blot 5 min in distilled water. Proceed with immunoprobing procedure (see Basic Protocol 2 and Alternate Protocol 4). Chemiluminescence Kit Developing the immunoblot 33 .Flow chart of Western blotting Electrophoresing the protein sample Assembling the Western blot sandwich Transferring proteins from gel to nitrocellulose paper Staining of transferred proteins (skip or not) Flow chart of Western blotting Blocking nonspecific antibody sites on the PVDF paper Probing electroblotted proteins with primary antibody Washing away nonspecifically bound primary antibody Detecting bound antibody by secondary antibody conjugated with horseradish peroxidase and Use an ECL .