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Western Blot: Technique, Theory, and Trouble Shooting

Western Blot: Technique, Theory, and Trouble Shooting Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot. Keywords: Bio-medical research, protein, western blot Address for correspondence: Dr. Ping-Chang Yang, Department of Pathology & Molecular Medicine, McMaster University, Room T3303, 50 Charlton Ave East, Hamilton, ON, Canada. E-mail: yangp@mcmaster.ca procedure, and in the later section, troubleshooting tips Introduction for common problems. Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins Technique is separated based on molecular weight, and thus by Cell lysis to extract protein type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each Protein can be extracted from different kind of samples, protein. The membrane is then incubated with labels such as tissue or cells. Below is the protocol to extract antibodies specific to the protein of interest. proteins from adherent cells. The unbound antibody is washed off leaving only the Adherent cells: bound antibody to the protein of interest. The bound 1. Wash cells in the tissue culture as fl k or dish by adding antibodies are then detected by developing the film. cold phosphate buffered saline (PBS) and rocking As the antibodies only bind to the protein of interest, gently. Discard PBS. (Tip: Keep tissue culture dish only one band should be visible. The thickness of the on ice throughout). band corresponds to the amount of protein present; thus 2. Add PBS and use a cell scraper to dislodge the cells. doing a standard can indicate the amount of protein Pipette the mixture into microcentrifuge tubes. present. The paper will first describe the protocol for 3. Centrifuge at 1500 RPM for 5 minutes and discard western blot, accompanied by pictures to help the the supernatant. reader and theory to rationalize the protocol. This 4. Add 180 µL of ice cold cell lysis buffer with 20 µL will be followed by the theoretical explanation of the fresh protease inhibitor cocktail. (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher Access this article online proportion of protease inhibitor cocktail). Quick Response Code: Website: 5. Incubate for 30 minutes on ice, and then clarify the www.najms.org lysate by spinning for 10 minutes at 12,000 RPM, at 4°C. 6. Transfer supernatant (or protein mix) to a fresh tube and store on ice or frozen at -20°C or -80°C. DOI: 10.4103/1947-2714.100998 7. Measure the concentration of protein using a spectrophotometer. North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | 429 Mahmood and Yang: Western blotting 4. Load marker (6 µL) followed by samples (15 µL) in Sample preparation to each well [Figure 4]. mass 1. Using, concentration = 5. Run the gel with low voltage (60 V) for separating volume gel; use higher voltage (140 V) for stacking gel determine the volume of protein extract to ensure 50 [Figure 5a and b]. µg in each well. 6. Run the gel for approximately an hour, or until the 2. Add 5 µL sample buffer to the sample, and make the dye front runs off the bottom of the gel [Figure 6]. volume in each lane equalized using double distilled H O (dd H O). Mix well. (Tip: Total volume of 15 µL 2 2 Electrotransfer per lane is suggested). 1. Cut 6 filter sheets to fit the measurement of the gel, 3. Heat the samples with dry plate for 5 minutes at and one polyvinylidene fluoride (PDVF) membrane 100ºC. with the same dimensions. 2. Wet the sponge and filter paper in transfer buffer, Gel preparation and wet the PDVF membrane in methanol. 3. Separate glass plates and retrieve the gel. 10% Stacking gel dd H O 3 mL 1 M Tris–HCl 2.1 mL 4. Create a transfer sandwich as follows: (pH 8.9) Sponge 30%Acr Bis 2.8 mL 3 Filter Papers 10%SDS 80 µL Gel 10%APS* 56 µL PVDF TEMED* 6 µL 3 Filter Papers 6% Separating gel dd H O 2 mL (Tip: Ensure there are no air bubbles between the gel 1M Tris–HCl 400 µL (pH 6.7) and PVDF membrane, and squeeze out extra liquid). 30%Acr Bis 600 µL 5. Relocat e the sandwich to the transfer apparatus, which 10%SDS* 36 µL should be placed on ice to maintain 4ºC. Add transfer 10%APS* 24 µL buffer to the apparatus, and ensure that the sandwich TEMED 4 µL is covered with the buffer. Place electrodes on top of *, APS: Ammonium Persulfate ; TEMED: Tetramethylethylenediamine; the sandwich, ensuring that the PVDF membrane is SDS: Sodium dodecyl sulfate between the gel and a positive electrode [Figure 7]. 6. Transfer for 90 minutes [Figure 8]. (Tip: The running 1. After preparing the 10% stacking gel solution, assemble time should be proportional to the thickness of the gel, the rack for gel solidification [Figure 1]. (Tip: 10% so this may be reduced to 45 minutes for 0.75 mm gels). AP and TEMED solidify the solution; therefore, both gels can be prepared at the same time, if the Blocking and antibody incubation abovementioned reagents are not added until the end). 1. Block the membrane with 5% skim milk in TBST* for 2. Add stacking gel solution carefully until the level 1 hour. is equal to the green bar holding the glass plates 2. Add primary antibody in 5% bovine serum albumin [Figure 2]. Add H O to the top. Wait for 15–30 minutes (BSA) and incubate overnight in 4ºC on a shaker until the gel turning solidified. (Tip: Using a suction [Figure 9]. pipette can make the process of adding the gel to the 3. Wash the membrane with TBST for 5 minutes. Do this glass plate easier). 3 times. (Tip: All washing and antibody incubation 3. Overlay the stacking gel with the separating gel, steps should be done on a shaker at room temperature after removing the water. (Tip: It is better to tilt the to ensure even agitation). apparatus and use a paper towel to remove the water). 4. Add secondary antibody in 5% skim milk in TBST, 4. Insert the comb, ensuring that there are no air bubbles. and incubate for 1 hour. 5. Wait until the gel is solidified. (Tip: Solidification can be 5. Wash the membrane with TBST for 5 minutes. Do easily checked by leaving some gel solution in a tube). this 3 times 6. Prepare ECL mix (following the proportion of Electrophoresis solution A and B provided by the manufacturer). 1. Pour the running buffer into the electrophorator Incubate the membrane for 1–2 minutes [Figure 10]. [Figure 3]. (Tip: Use a 1000 µL pipette to ensure that ECL covers 2. Place gel inside the electrophorator and connect to a the top and bottom of the membrane). power supply. (Tip: When connecting to the power 7. Visualize the result in the dark room [Figure 11]. (Tip: source always connect red to red, and black to black). If the background is too strong, reduce exposure time). 3. Make sure buffer covers the gel completely, and remove the comb carefully. *, TBST: Tris-Buffered Saline Tween-20 430 North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | Mahmood and Yang: Western blotting Figure 1: Assembled rack for gel solidification Figure 2: Add gel solution using a transfer pipette Figure 3: Add running buffer to the electrophorator Recipe 1. Dissolve the following in 800 ml of distilled H O • 8.8 g of NaCl Figure 4: Add samples and molecular marker to the gel, after • 0.2g of KCl removing the combs • 3g of Tris base 2. Add 500ul of Tween-20 is often measured using a spectrophotometer. Using this 3. Adjust the pH to 7.4 concentration allows to measure the mass of the protein 4. Add distilled H O to 1L that is being loaded into each well by the relationship 5. Sterilize by filtration or autoclaving between concentration, mass, and volume. Theory After determining the appropriate volume of the sample, it is diluted into a loading buffer, which contains glycerol Sample preparation so that the samples sink easily into the wells of the gel. Cell lysates are the most common form of sample used A tracking dye (bromophenol blue) is also present in the for western blot. Protein extraction attempts to collect all buffer allowing the researcher to see how far the separation the proteins in the cell cytosol. This should be done in has progressed. The sample is heated after being diluted a cold temperature with protease inhibitors to prevent into a loading buffer, in order to denature the higher order denaturing of the proteins. Since tissue sample display structure, while retaining suld fi e bridges. Denaturing the a higher degree of structure, mechanical invention, such high structure ensures that the negative charge of amino as homogenization, or sonication is needed to extract acids is not neutralized, enabling the protein to move in the proteins. an electric field (applied during electrotransfer). After extracting the protein, it is very important to have a good idea of the extract’s concentration. This eventually It is also very important to have positive and negative allows the researcher to ensure that the samples are being controls for the sample. For a positive control a known compared on an equivalent basis. Protein concentration source of target protein, such as purified protein or a North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | 431 Mahmood and Yang: Western blotting a b Figure 5: (a) Samples running through the stacking gel (lower voltage). (b): Samples running through the separating gel (higher voltage) Figure 6: Run the gel to the bottom of the electrophorator Figure 7: Transfer should be done on ice Figure 8: Membrane after transfer control lysate is used. This helps to confirm the identity Figure 9: Use a shaker to incubate the membrane with antibody of the protein, and the activity of the antibody. A negative control is a null cell line, such as β-actin, is used as well defined bands. The lower gel, called the separating, to confirm that the staining is not nonspecific. or resolving gel, is basic (pH 8.8), and has a higher polyacrylamide content, making the gel’s pores Gel electrophoresis narrower. Protein is thus separated by their size more so Western blot uses two different types of agarose gel: in this gel, as the smaller proteins to travel more easily, stacking and separating gel. The higher, stacking gel and hence rapidly, than larger proteins. is slightly acidic (pH 6.8) and has a lower acrylamide concentration making a porous gel, which separates The proteins when loaded on the gel have a negative protein poorly but allows them to form thin, sharply charge, as they have been denatured by heating, and will 432 North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | Mahmood and Yang: Western blotting travel toward the positive electrode when a voltage is applied. Gels are usually made by pouring them between two glass or plastic plates, using the solution described in the protocol section. The samples and a marker are loaded into the wells, and the empty wells are loaded with sample buffer. The gel is then connected to the power supply and allowed to run. The voltage is very important, as a high voltage can overheat and distort the bands. Blotting After separating the protein mixture, it is transferred to a membrane. The transfer is done using an electric field oriented perpendicular to the surface of the gel, causing proteins to move out of the gel and onto the membrane. The membrane is placed between the gel Figure 10: Incubate the membrane with ECL mix using a 1000 µL surface and the positive electrode in a sandwich. The pipette to help the process sandwich includes a fiber pad (sponge) at each end, and filter papers to protect the gel and blotting membrane [Figure 12]. Here two things are very important: (1) the close contact of gel and membrane to ensure a clear image and (2) the placement of the membrane between the gel and the positive electrode. The membrane must be placed as such, so that the negatively charged proteins can migrate from the gel to the membrane. This type of transfer is called electrophoretic transfer, and can be done in semi-dry or wet conditions. Wet conditions are usually more reliable as it is less likely to dry out the gel, and is preferred for larger proteins. Figure 11: Use the cassette to expose the membrane in the dark room The membrane, the solid support, is an essential part of this process. There are two types of membrane: nitrocellulose and PVDF. Nitrocellulose is used for its high affinity for protein and its retention abilities. However, it is brittle, and does not allow the membrane to be used for reprobing. In this regard, PVDF membranes provide better mechanical support and allow the blot to be reprobed and stored. However, the background is higher in the PVDF membranes and therefore, washing carefully is very important. Washing , blocking and antibody incubation Blocking is a very important step of western blotting, as it prevents antibodies from binding to the membrane Figure 12: Assembly of a sandwich in western Blot nonspecifically. Blocking is often made with 5% BSA or nonfat dried milk diluted in TBST to reduce the background. higher amounts than the secondary antibody. Putting it in BSA solution allows the antibody to be reused, if the Nonfat dried milk is often preferred as it is inexpensive blot does not give good result. and widely available. However, milk proteins are not compatible with all detection labels, so care must be The concentration of the antibody depends on the taken to choose the appropriate blocking solution. For instruction by the manufacturer. The antibody can be example, BSA blocking solutions are preferred with diluted in a wash buffer, such as PBS or TBST. Washing biotin and AP antibody labels, and antiphosphoprotein antibodies, since milk contains casein, which is itself a is very important as it minimized background and phosphoprotein and biotin, thus interfering with the removes unbound antibody. However, the membrane assay results. It is often a good strategy to incubate the should not be left to wash for a really long time, as it can primary antibody with BSA since it is usually needed in also reduce the signal. North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | 433 Mahmood and Yang: Western blotting The membrane is then detected using the label antibody, Another reason for no visible bands is the lowest usually with an enzyme such as horseradish peroxidase concentration or absence of the antigen. In this case, (HRP), which is detected by the signal it produces antigen from another source can be used to confirm corresponding to the position of the target protein. This whether the problem lies with the sample or with other signal is captured on a film which is usually developed elements, such as the antibody. Moreover, prolonged washing can also decrease the signal. Buffers can also in a dark room. contribute to the problem. It should be ensured that buffers like the transfer buffer, TBST, running buffer Quantification and ECL are all new and noncontaminated. If the It is very important to be aware that the data produced buffers are contaminated with sodium azide, it can with a western blot is typically considered to be semi- inactivate HRP. quantitative. This is because it provides a relative comparison of protein levels, but not an absolute Similarly, weak signals can be caused by low concentration measure of quantity. There are two reasons for this; of antibody or antigen. Increasing exposure time can also first, there are variations in loading and transfer rates help to make the band clearer. Another reason could be between the samples in separate lanes which are different nonfat dry milk masking the antigen. In this case use on separate blots. These differences will need to be BSA or decrease the amount of milk used. standardized before a more precise comparison can be made. Second, the signal generated by detection is not High background is often caused by too high concentration linear across the concentration range of samples. Thus, of the antibody, which can bind to PVDF membranes. since the signal produced is not linear, it should not be Another problem could be the buffers, which may be used to model the concentration. too old. Increasing the washing time can also help to decrease the background. Additionally, too high of an Troubleshooting exposure can also lead to this problem. Therefore, it is Even though the procedure for western blot is simple, advisable to check different exposure times to achieve many problems can arise, leading to unexpected results. an optimum time. The problem can be grouped into five categories: (1) unusual or unexpected bands, (2) no bands, (3) faint Patchy and uneven spots on the blot are usually caused bands or weak signal, (4) high background on the blot, by improper transfer. If there are air bubbles trapped and (5) patchy or uneven spots on the blot. between the gel and the membrane, it will appear darker on the film. It is also important to use a shaker for all Unusual or unexpected bands can be due to protease incubation, so that there is no uneven agitation during the degradation, which produces bands at unexpected incubation. Once again, washing is of utmost importance positions. In this case it is advisable to use a fresh sample as well to wash the background. This problem can also which had been kept on ice or alter the antibody. If be caused by antibodies binding to the blocking agents; the protein seems to be in too high of a position, then in this case another blocking agent should be tried. reheating the sample can help to break the quaternary Filtering the blocking agent can also help to remove some protein structure. Similarly, blurry bands are often contaminants. Finally, this problem can also be caused by aggregation of the secondary antibody; in this case, the caused by high voltage or air bubbles present during secondary antibody should be centrifuged and filtered transfer. In this case, it should be ensured that the gel is to remove the aggregated. run at a lower voltage, and that the transfer sandwich is prepared properly. In addition, changing the running buffer can also help the problem. Nonflat bands can be Conclusion the result of too fast of a travel through the gel, due to low resistance. To fix this the gel should be optimized Western blot is a technique that is very useful for protein to fit the sample. Finally, white (negative) bands on the detection as it allows the user to quantify the protein film are due to too much protein or antibody. expression as well. This paper covered the protocol, the theory behind that protocol, and some troubleshooting techniques. Western blot can be seen as an intricate Another problem: no bands can also arise due to many balance, as the researcher attempts to get a nonspecific, reasons related to antibody, antigen, or buffer used. yet strong signal. If an improper antibody is used, either primary or secondary, the band will not show. In addition, the concentration of the antibody should be appropriate How to cite this article: Mahmood T, Yang P. Western blot: Technique, as well; if the concentration is too low, the signal theory, and trouble shooting. North Am J Med Sci 2012;4:429-34. may not be visible. It is important to remember that Source of Support: Nil. Conflict of Interest: None declared. some antibodies are not to be used for western blot. 434 North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png North American Journal of Medical Sciences Pubmed Central

Western Blot: Technique, Theory, and Trouble Shooting

North American Journal of Medical Sciences , Volume 4 (9) – Sep 1, 2012

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Pubmed Central
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Copyright: © North American Journal of Medical Sciences
ISSN
2250-1541
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1947-2714
DOI
10.4103/1947-2714.100998
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Abstract

Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot. Keywords: Bio-medical research, protein, western blot Address for correspondence: Dr. Ping-Chang Yang, Department of Pathology & Molecular Medicine, McMaster University, Room T3303, 50 Charlton Ave East, Hamilton, ON, Canada. E-mail: yangp@mcmaster.ca procedure, and in the later section, troubleshooting tips Introduction for common problems. Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins Technique is separated based on molecular weight, and thus by Cell lysis to extract protein type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each Protein can be extracted from different kind of samples, protein. The membrane is then incubated with labels such as tissue or cells. Below is the protocol to extract antibodies specific to the protein of interest. proteins from adherent cells. The unbound antibody is washed off leaving only the Adherent cells: bound antibody to the protein of interest. The bound 1. Wash cells in the tissue culture as fl k or dish by adding antibodies are then detected by developing the film. cold phosphate buffered saline (PBS) and rocking As the antibodies only bind to the protein of interest, gently. Discard PBS. (Tip: Keep tissue culture dish only one band should be visible. The thickness of the on ice throughout). band corresponds to the amount of protein present; thus 2. Add PBS and use a cell scraper to dislodge the cells. doing a standard can indicate the amount of protein Pipette the mixture into microcentrifuge tubes. present. The paper will first describe the protocol for 3. Centrifuge at 1500 RPM for 5 minutes and discard western blot, accompanied by pictures to help the the supernatant. reader and theory to rationalize the protocol. This 4. Add 180 µL of ice cold cell lysis buffer with 20 µL will be followed by the theoretical explanation of the fresh protease inhibitor cocktail. (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher Access this article online proportion of protease inhibitor cocktail). Quick Response Code: Website: 5. Incubate for 30 minutes on ice, and then clarify the www.najms.org lysate by spinning for 10 minutes at 12,000 RPM, at 4°C. 6. Transfer supernatant (or protein mix) to a fresh tube and store on ice or frozen at -20°C or -80°C. DOI: 10.4103/1947-2714.100998 7. Measure the concentration of protein using a spectrophotometer. North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | 429 Mahmood and Yang: Western blotting 4. Load marker (6 µL) followed by samples (15 µL) in Sample preparation to each well [Figure 4]. mass 1. Using, concentration = 5. Run the gel with low voltage (60 V) for separating volume gel; use higher voltage (140 V) for stacking gel determine the volume of protein extract to ensure 50 [Figure 5a and b]. µg in each well. 6. Run the gel for approximately an hour, or until the 2. Add 5 µL sample buffer to the sample, and make the dye front runs off the bottom of the gel [Figure 6]. volume in each lane equalized using double distilled H O (dd H O). Mix well. (Tip: Total volume of 15 µL 2 2 Electrotransfer per lane is suggested). 1. Cut 6 filter sheets to fit the measurement of the gel, 3. Heat the samples with dry plate for 5 minutes at and one polyvinylidene fluoride (PDVF) membrane 100ºC. with the same dimensions. 2. Wet the sponge and filter paper in transfer buffer, Gel preparation and wet the PDVF membrane in methanol. 3. Separate glass plates and retrieve the gel. 10% Stacking gel dd H O 3 mL 1 M Tris–HCl 2.1 mL 4. Create a transfer sandwich as follows: (pH 8.9) Sponge 30%Acr Bis 2.8 mL 3 Filter Papers 10%SDS 80 µL Gel 10%APS* 56 µL PVDF TEMED* 6 µL 3 Filter Papers 6% Separating gel dd H O 2 mL (Tip: Ensure there are no air bubbles between the gel 1M Tris–HCl 400 µL (pH 6.7) and PVDF membrane, and squeeze out extra liquid). 30%Acr Bis 600 µL 5. Relocat e the sandwich to the transfer apparatus, which 10%SDS* 36 µL should be placed on ice to maintain 4ºC. Add transfer 10%APS* 24 µL buffer to the apparatus, and ensure that the sandwich TEMED 4 µL is covered with the buffer. Place electrodes on top of *, APS: Ammonium Persulfate ; TEMED: Tetramethylethylenediamine; the sandwich, ensuring that the PVDF membrane is SDS: Sodium dodecyl sulfate between the gel and a positive electrode [Figure 7]. 6. Transfer for 90 minutes [Figure 8]. (Tip: The running 1. After preparing the 10% stacking gel solution, assemble time should be proportional to the thickness of the gel, the rack for gel solidification [Figure 1]. (Tip: 10% so this may be reduced to 45 minutes for 0.75 mm gels). AP and TEMED solidify the solution; therefore, both gels can be prepared at the same time, if the Blocking and antibody incubation abovementioned reagents are not added until the end). 1. Block the membrane with 5% skim milk in TBST* for 2. Add stacking gel solution carefully until the level 1 hour. is equal to the green bar holding the glass plates 2. Add primary antibody in 5% bovine serum albumin [Figure 2]. Add H O to the top. Wait for 15–30 minutes (BSA) and incubate overnight in 4ºC on a shaker until the gel turning solidified. (Tip: Using a suction [Figure 9]. pipette can make the process of adding the gel to the 3. Wash the membrane with TBST for 5 minutes. Do this glass plate easier). 3 times. (Tip: All washing and antibody incubation 3. Overlay the stacking gel with the separating gel, steps should be done on a shaker at room temperature after removing the water. (Tip: It is better to tilt the to ensure even agitation). apparatus and use a paper towel to remove the water). 4. Add secondary antibody in 5% skim milk in TBST, 4. Insert the comb, ensuring that there are no air bubbles. and incubate for 1 hour. 5. Wait until the gel is solidified. (Tip: Solidification can be 5. Wash the membrane with TBST for 5 minutes. Do easily checked by leaving some gel solution in a tube). this 3 times 6. Prepare ECL mix (following the proportion of Electrophoresis solution A and B provided by the manufacturer). 1. Pour the running buffer into the electrophorator Incubate the membrane for 1–2 minutes [Figure 10]. [Figure 3]. (Tip: Use a 1000 µL pipette to ensure that ECL covers 2. Place gel inside the electrophorator and connect to a the top and bottom of the membrane). power supply. (Tip: When connecting to the power 7. Visualize the result in the dark room [Figure 11]. (Tip: source always connect red to red, and black to black). If the background is too strong, reduce exposure time). 3. Make sure buffer covers the gel completely, and remove the comb carefully. *, TBST: Tris-Buffered Saline Tween-20 430 North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | Mahmood and Yang: Western blotting Figure 1: Assembled rack for gel solidification Figure 2: Add gel solution using a transfer pipette Figure 3: Add running buffer to the electrophorator Recipe 1. Dissolve the following in 800 ml of distilled H O • 8.8 g of NaCl Figure 4: Add samples and molecular marker to the gel, after • 0.2g of KCl removing the combs • 3g of Tris base 2. Add 500ul of Tween-20 is often measured using a spectrophotometer. Using this 3. Adjust the pH to 7.4 concentration allows to measure the mass of the protein 4. Add distilled H O to 1L that is being loaded into each well by the relationship 5. Sterilize by filtration or autoclaving between concentration, mass, and volume. Theory After determining the appropriate volume of the sample, it is diluted into a loading buffer, which contains glycerol Sample preparation so that the samples sink easily into the wells of the gel. Cell lysates are the most common form of sample used A tracking dye (bromophenol blue) is also present in the for western blot. Protein extraction attempts to collect all buffer allowing the researcher to see how far the separation the proteins in the cell cytosol. This should be done in has progressed. The sample is heated after being diluted a cold temperature with protease inhibitors to prevent into a loading buffer, in order to denature the higher order denaturing of the proteins. Since tissue sample display structure, while retaining suld fi e bridges. Denaturing the a higher degree of structure, mechanical invention, such high structure ensures that the negative charge of amino as homogenization, or sonication is needed to extract acids is not neutralized, enabling the protein to move in the proteins. an electric field (applied during electrotransfer). After extracting the protein, it is very important to have a good idea of the extract’s concentration. This eventually It is also very important to have positive and negative allows the researcher to ensure that the samples are being controls for the sample. For a positive control a known compared on an equivalent basis. Protein concentration source of target protein, such as purified protein or a North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | 431 Mahmood and Yang: Western blotting a b Figure 5: (a) Samples running through the stacking gel (lower voltage). (b): Samples running through the separating gel (higher voltage) Figure 6: Run the gel to the bottom of the electrophorator Figure 7: Transfer should be done on ice Figure 8: Membrane after transfer control lysate is used. This helps to confirm the identity Figure 9: Use a shaker to incubate the membrane with antibody of the protein, and the activity of the antibody. A negative control is a null cell line, such as β-actin, is used as well defined bands. The lower gel, called the separating, to confirm that the staining is not nonspecific. or resolving gel, is basic (pH 8.8), and has a higher polyacrylamide content, making the gel’s pores Gel electrophoresis narrower. Protein is thus separated by their size more so Western blot uses two different types of agarose gel: in this gel, as the smaller proteins to travel more easily, stacking and separating gel. The higher, stacking gel and hence rapidly, than larger proteins. is slightly acidic (pH 6.8) and has a lower acrylamide concentration making a porous gel, which separates The proteins when loaded on the gel have a negative protein poorly but allows them to form thin, sharply charge, as they have been denatured by heating, and will 432 North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | Mahmood and Yang: Western blotting travel toward the positive electrode when a voltage is applied. Gels are usually made by pouring them between two glass or plastic plates, using the solution described in the protocol section. The samples and a marker are loaded into the wells, and the empty wells are loaded with sample buffer. The gel is then connected to the power supply and allowed to run. The voltage is very important, as a high voltage can overheat and distort the bands. Blotting After separating the protein mixture, it is transferred to a membrane. The transfer is done using an electric field oriented perpendicular to the surface of the gel, causing proteins to move out of the gel and onto the membrane. The membrane is placed between the gel Figure 10: Incubate the membrane with ECL mix using a 1000 µL surface and the positive electrode in a sandwich. The pipette to help the process sandwich includes a fiber pad (sponge) at each end, and filter papers to protect the gel and blotting membrane [Figure 12]. Here two things are very important: (1) the close contact of gel and membrane to ensure a clear image and (2) the placement of the membrane between the gel and the positive electrode. The membrane must be placed as such, so that the negatively charged proteins can migrate from the gel to the membrane. This type of transfer is called electrophoretic transfer, and can be done in semi-dry or wet conditions. Wet conditions are usually more reliable as it is less likely to dry out the gel, and is preferred for larger proteins. Figure 11: Use the cassette to expose the membrane in the dark room The membrane, the solid support, is an essential part of this process. There are two types of membrane: nitrocellulose and PVDF. Nitrocellulose is used for its high affinity for protein and its retention abilities. However, it is brittle, and does not allow the membrane to be used for reprobing. In this regard, PVDF membranes provide better mechanical support and allow the blot to be reprobed and stored. However, the background is higher in the PVDF membranes and therefore, washing carefully is very important. Washing , blocking and antibody incubation Blocking is a very important step of western blotting, as it prevents antibodies from binding to the membrane Figure 12: Assembly of a sandwich in western Blot nonspecifically. Blocking is often made with 5% BSA or nonfat dried milk diluted in TBST to reduce the background. higher amounts than the secondary antibody. Putting it in BSA solution allows the antibody to be reused, if the Nonfat dried milk is often preferred as it is inexpensive blot does not give good result. and widely available. However, milk proteins are not compatible with all detection labels, so care must be The concentration of the antibody depends on the taken to choose the appropriate blocking solution. For instruction by the manufacturer. The antibody can be example, BSA blocking solutions are preferred with diluted in a wash buffer, such as PBS or TBST. Washing biotin and AP antibody labels, and antiphosphoprotein antibodies, since milk contains casein, which is itself a is very important as it minimized background and phosphoprotein and biotin, thus interfering with the removes unbound antibody. However, the membrane assay results. It is often a good strategy to incubate the should not be left to wash for a really long time, as it can primary antibody with BSA since it is usually needed in also reduce the signal. North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 | 433 Mahmood and Yang: Western blotting The membrane is then detected using the label antibody, Another reason for no visible bands is the lowest usually with an enzyme such as horseradish peroxidase concentration or absence of the antigen. In this case, (HRP), which is detected by the signal it produces antigen from another source can be used to confirm corresponding to the position of the target protein. This whether the problem lies with the sample or with other signal is captured on a film which is usually developed elements, such as the antibody. Moreover, prolonged washing can also decrease the signal. Buffers can also in a dark room. contribute to the problem. It should be ensured that buffers like the transfer buffer, TBST, running buffer Quantification and ECL are all new and noncontaminated. If the It is very important to be aware that the data produced buffers are contaminated with sodium azide, it can with a western blot is typically considered to be semi- inactivate HRP. quantitative. This is because it provides a relative comparison of protein levels, but not an absolute Similarly, weak signals can be caused by low concentration measure of quantity. There are two reasons for this; of antibody or antigen. Increasing exposure time can also first, there are variations in loading and transfer rates help to make the band clearer. Another reason could be between the samples in separate lanes which are different nonfat dry milk masking the antigen. In this case use on separate blots. These differences will need to be BSA or decrease the amount of milk used. standardized before a more precise comparison can be made. Second, the signal generated by detection is not High background is often caused by too high concentration linear across the concentration range of samples. Thus, of the antibody, which can bind to PVDF membranes. since the signal produced is not linear, it should not be Another problem could be the buffers, which may be used to model the concentration. too old. Increasing the washing time can also help to decrease the background. Additionally, too high of an Troubleshooting exposure can also lead to this problem. Therefore, it is Even though the procedure for western blot is simple, advisable to check different exposure times to achieve many problems can arise, leading to unexpected results. an optimum time. The problem can be grouped into five categories: (1) unusual or unexpected bands, (2) no bands, (3) faint Patchy and uneven spots on the blot are usually caused bands or weak signal, (4) high background on the blot, by improper transfer. If there are air bubbles trapped and (5) patchy or uneven spots on the blot. between the gel and the membrane, it will appear darker on the film. It is also important to use a shaker for all Unusual or unexpected bands can be due to protease incubation, so that there is no uneven agitation during the degradation, which produces bands at unexpected incubation. Once again, washing is of utmost importance positions. In this case it is advisable to use a fresh sample as well to wash the background. This problem can also which had been kept on ice or alter the antibody. If be caused by antibodies binding to the blocking agents; the protein seems to be in too high of a position, then in this case another blocking agent should be tried. reheating the sample can help to break the quaternary Filtering the blocking agent can also help to remove some protein structure. Similarly, blurry bands are often contaminants. Finally, this problem can also be caused by aggregation of the secondary antibody; in this case, the caused by high voltage or air bubbles present during secondary antibody should be centrifuged and filtered transfer. In this case, it should be ensured that the gel is to remove the aggregated. run at a lower voltage, and that the transfer sandwich is prepared properly. In addition, changing the running buffer can also help the problem. Nonflat bands can be Conclusion the result of too fast of a travel through the gel, due to low resistance. To fix this the gel should be optimized Western blot is a technique that is very useful for protein to fit the sample. Finally, white (negative) bands on the detection as it allows the user to quantify the protein film are due to too much protein or antibody. expression as well. This paper covered the protocol, the theory behind that protocol, and some troubleshooting techniques. Western blot can be seen as an intricate Another problem: no bands can also arise due to many balance, as the researcher attempts to get a nonspecific, reasons related to antibody, antigen, or buffer used. yet strong signal. If an improper antibody is used, either primary or secondary, the band will not show. In addition, the concentration of the antibody should be appropriate How to cite this article: Mahmood T, Yang P. Western blot: Technique, as well; if the concentration is too low, the signal theory, and trouble shooting. North Am J Med Sci 2012;4:429-34. may not be visible. It is important to remember that Source of Support: Nil. Conflict of Interest: None declared. some antibodies are not to be used for western blot. 434 North American Journal of Medical Sciences | September 2012 | Volume 4 | Issue 9 |

Journal

North American Journal of Medical SciencesPubmed Central

Published: Sep 1, 2012

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