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western blot upd.pptx

  1. Western Blot
  2. Introduction Western blotting (WB) or immunoblot is an antibody-based experimental technique used to detect and quantify target proteins, which are often within a complex mixture extracted from cells or tissue ● WB involves multiple steps for detection of different proteins, there is no one particular set of optimal conditions suitable for all proteins. ● Researchers usually spend considerable time optimizing the conditions to obtain the best signal-to-noise ratios, yet difficulties persist in obtaining consistent and high-quality results1,2
  3. Principles WB includes the following steps: 1. Proteins are separated from the mixture by sodium dodecyl sulfate (SDS)– polyacrylamide gel electrophoresis according to their molecular weights 2. The separated proteins are transferred and bound to a solid membrane 3. The target protein on the membrane is detected by the immunological method 4. The identification of a specific protein is based on two parameters: molecular weight and signal intensity. Molecular weight could be estimated by prestained molecular weight markers. The signal is determined by a secondary antibody following the addition of primary antibodies to detect the protein blotted onto the membrane1,2
  4. Four step Western blot procedure5
  5. Flowchart of Western blot procedure. The main steps of the basic protocol with one and two-step probing are shown3
  6. Sample preparation 1. The first step of WB is sample preparation. It requires to lyse the cells, whichin case of tissues is preceded by mechanical or enzymatic homogenization, to extract and solubilize proteins 2. Depending on the cellular structures (nucleus, cytoplasm, plasma membrane, etc.) and cell type (e.g., mammalian cells, bacteria, yeast), different lysis buffer might be used 3. Typically, it is composed of a buffering agent (e.g., Tris–HCl), salts such as sodium chloride, detergent disrupting lipids, and protease inhibitors to prevent protein degradation1,2
  7. Electrophoresis 1. Samples are then subjected to polyacrylamide gel electrophoresis (PAGE), which separates proteins either based on their structure and isoelectric point (native-PAGE) or their size (SDS-PAGE) 2. Proteins need to possess a negative charge to migrate through the gel pores when subjected to an electromagnetic field. 3. In native-PAGE either the intrinsic charge of a target protein or Coomasie G-250 give proteins a negative charge. In SDS-PAGE, all proteins are negatively charged due to the binding of SDS1,2
  8. Schematic of SDS-Page electrophoresis 1. Polyacrylamide two-part gel composed of a stacking (top) and a resolving (bottom) gel is enclosed between two glass plates and submerged in running buffer. 2. Protein samples denatured with SDS are pipetted into wells on top of the gel. 3. Application of electromagnetic field causes the migration of proteins toward the anode (+) resulting in their separation based on the molecular weight.1,2
  9. Electrotransfer 1. Proteins separated by PAGE need to be transferred from the gel to a membrane by electrophoretic transfer (or electrotransfer) for further processing 2. A blot sandwich is prepared where a membrane is tightly touching the gel and sandwiched between filter paper (or similar support) in a transfer buffer 3. It is important to ensure a good contact between the gel and the membrane as air bubbles would prevent the transfer 4. An electromagnetic field is applied perpendicularly to the gel surface to transfer the proteins from the gel to the membrane.1,,2,3
  10. Electrotransfer method There are two main methods of electrotransfer: wet and semidry No. Wet transfer Semidry transfer 1 Wet transfer: the tight contact between the gel and the membrane during the electrotransfer is ensured by solid support and the sandwich is entirely submerged in the transfer buffer Semidry transfer: the sandwich is placed directly between electrodes and only the filter paper is soaked with the transfer buffer 2 Wet transfer is more flexible to optimization (e.g., time, voltage) and with proper cooling may be run overnight resulting in more complete transfer semidry method has low buffering capacity and cannot be run for prolonged time due to the risk of drying 3 allows the transfer of broader range of protein size and is favorable for big proteins (>100 kDa) the transfer is less effective and may result in difficulties of detecting the target, especially if the protein has a high molecular weight and/or is not abundant 4 it takes much longer (at least 1 hour) and requires greater buffer volumes it is very quick (10 minutes to an hour) and many membranes can be processed at the same time with minimal use of buffer.3
  11. Three types of western blot7
  12. Example of Western Blot with wet-transfer system4
  13. Electrotransfer Membrane There are two main types of membranes commonly used: nitrocellulose and PVDF (polyvinylidene difluoride) Buffer Composition The most common transfer buffers are based on Tris and glycine solutions supplemented with methanol and SDS.3
  14. Blocking 1. Membranes used in electro transfer are characterized by high protein binding capabilities and therefore the surface unoccupied by transferred proteins requires blocking to reduce background 2. Ideal blocking solution needs to be optimized but in general 1–5% nonfat dry milk (NFDM) or bovine serum albumin (BSA) solutions have proved to be successful. 3
  15. Probing a. The membrane needs to be probed with an antibody recognizing the protein of interest b. This step is crucial and may require extensive optimization as each antibody will have different strength and specificity c. Probing can be performed in one or two steps: 1. the one-step probing: the antibody against the protein of interest is conjugated with an enzyme (such as horseradish peroxidase, HRP, or alkaline phosphatase, AP) or a fluorophore allowing its detections 2. the two-step probing: two different antibodies are used: the primary antibody against the protein of interest and a secondary, conjugated antibody against the primary antibody.3
  16. Washings 1. Removal of the background and ability to detect the specific signal 2. Usually, a very mild detergent solution is used for washings.3
  17. Detection 1. The most popular method is using antibodies conjugated with HRP and AP enzymes 2. The antibody can also be conjugated with fluorophores such as fluorescein (FITC) or rhodamine (TRITC) allowing direct visualization of the antibody using fluorescence. 3
  18. Stripping (optional) 1. The stripping may not be consistent throughout the whole membrane 2. Therefore, a re-probed blot should not be used for semi quantification and the result should be interpreted with caution. 3
  19. Applications 1. The main research application of the WB assay is to detect the presence of a protein of interest in a variety of systems. It can be used to determine expression of a given protein in organelles, cells, tissues, or embryos/whole organism 2. Both endogenous and heterologously expressed proteins can be detected 3. Assessment of posttranslational modification of the target protein such as phosphorylation (with phospho-specific antibodies), ubiquitination, or sumoylation detected with a combination of WB with immunoprecipitation (IP) 4. WB is used to detect the prion protein causing bovine spongiform encephalopathy, bovine spongiform encephalopathy (BSE) in cows 5. In medicine, it is often used as a confirmatory test [usually in combination with enzyme- linked immunosorbent assay (ELISA)] in diagnosis of several diseases such as lyme disease or HIV. 1,2,3
  20. Selecting a secondary antibody, both the type of primary antibody and the requirements of subsequent detection schemes 1. Species source of the primary antibody: the reactivity of the secondary antibody should be consistent with the species source of the primary antibody used 2. Type of primary antibody: the secondary antibody must match the class or subclass of the primary antibody. 3. Species source of the secondary antibody: there is usually no predictable connection between species source and the quality of the secondary antibody. However, the use of secondary antibodies from the same species as the primary antibodies should be avoided, especially in double-labeling experiments 4. Coupling of probes to the secondary antibody: probes coupled to secondary antibodies mainly include enzymes (such as horseradish peroxidase and alkaline phosphatase), fluorescent molecules (FITC, rhodamine, Texas Red, PE, Dylight, etc.), biotin, and gold particles.
  21. Selecting a secondary antibody.. (part 2) 5. Another critical issue is the selection of the loading control, which has been widely used in the normalization of WB results to adjust for systematic differences between samples or even between experiments 6. Moreover, some housekeeping proteins are also used as markers for subcellular compartments according to their intracellular distribution.
  22. Markers for subcellular compartments
  23. Therefore, to achieve reproducible WB results, the following information should be provided in “Materials and Methods” of a paper: 1. The primary antibody species (for monoclonal or polyclonal antibodies), isotype (IgG, IgY, etc.), and epitopes generated 2. Secondary antibody species, isotype, and labeling 3. Source of the primary and secondary antibodies; catalog and lot numbers are needed if they were obtained from a commercial company 4. Dilution and incubation conditions of the primary and secondary antibodies. 5. Type of blotting membrane (nitrocellulose, polyvinylidene fluoride, etc.) 6. Blocking agents (bovine serum albumin (0.2%–5.0%), nonfat milk, casein, gelatin, etc.)1,2,3
  24. Schematic of western blotting1,2 process
  25. Representative WB bands1,2
  26. KEY LIMITATIONS 1. WB is not a quantitative technique as it will not tell how much of the protein is expressed 2. WB in general is quite sensitive, down to the low femtogram range in the best cases. In reality, however, due to accumulation of intrinsic inefficiencies of the technique (such as transfer, recognition by antibodies, etc.) the detection limit is usually higher 3. WB is as specific as the antibody used for the membrane probing and therefore in some cases when the specificity of the antibody is poor the result may be not easy to interpret 4. As in any multistep technique, there are many steps that can go wrong resulting in poor or no interpretable results.3
  27. Glossary2