Western Blot Wash and Reblot Again

• Journal List
• Due north Am J Med Sci
• 5.four(9); 2012 Sep
• PMC3456489

N Am J Med Sci. 2012 Sep; 4(9): 429–434.

Western Blot: Technique, Theory, and Trouble Shooting

Tahrin Mahmood

Department of Pathology and Molecular Medicine, McMaster Academy, Hamilton, ON, Canada

Ping-Chang Yang

Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada

Abstract

Western blotting is an of import technique used in cell and molecular biological science. By using a western absorb, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses 3 elements to reach this task: (ane) separation past size, (ii) transfer to a solid support, and (iii) marking target protein using a proper master and secondary antibody to visualize. This newspaper will try to explain the technique and theory behind western absorb, and offer some ways to troubleshoot.

Keywords: Bio-medical enquiry, protein, western blot

Introduction

Western blot is often used in enquiry to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each poly peptide. The membrane is then incubated with labels antibodies specific to the protein of interest.

The unbound antibiotic is washed off leaving only the bound antibody to the protein of interest. The jump antibodies are then detected past developing the film. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the ring corresponds to the corporeality of protein nowadays; thus doing a standard can indicate the amount of protein present. The paper volition start describe the protocol for western blot, accompanied by pictures to assist the reader and theory to rationalize the protocol. This will be followed by the theoretical explanation of the procedure, and in the later on section, troubleshooting tips for common problems.

Technique

Cell lysis to extract protein

Protein can be extracted from different kind of samples, such equally tissue or cells. Beneath is the protocol to extract proteins from adherent cells.

Adherent cells:

1. Launder cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS. (Tip: Keep tissue culture dish on water ice throughout).

2. Add PBS and utilise a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes.

3. Centrifuge at 1500 RPM for 5 minutes and discard the supernatant.

4. Add together 180 μL of ice cold jail cell lysis buffer with twenty μL fresh protease inhibitor cocktail. (Tip: If protein concentration is not loftier enough at the end, it is brash to repeat the process with a higher proportion of protease inhibitor cocktail).

5. Incubate for xxx minutes on ice, and then clarify the lysate by spinning for 10 minutes at 12,000 RPM, at iv°C.

6. Transfer supernatant (or poly peptide mix) to a fresh tube and store on ice or frozen at -xx°C or -80°C.

7. Mensurate the concentration of protein using a spectrophotometer.

Sample preparation

1. 

   decide the volume of poly peptide extract to ensure fifty μg in each well.

2. Add 5 μL sample buffer to the sample, and make the book in each lane equalized using double distilled H₂O (dd H_twoO). Mix well. (Tip: Full book of 15 μL per lane is suggested).

3. Rut the samples with dry plate for v minutes at 100°C.

Gel preparation

1. After preparing the ten% stacking gel solution, assemble the rack for gel solidification [Figure ane]. (Tip: ten% AP and TEMED solidify the solution; therefore, both gels tin exist prepared at the same time, if the abovementioned reagents are non added until the cease).

   

   Assembled rack for gel solidification

2. Add stacking gel solution carefully until the level is equal to the green bar property the glass plates [Figure 2]. Add H_iiO to the top. Wait for xv–thirty minutes until the gel turning solidified. (Tip: Using a suction pipette tin can make the process of adding the gel to the drinking glass plate easier).

   

   Add gel solution using a transfer pipette

3. Overlay the stacking gel with the separating gel, after removing the water. (Tip: It is meliorate to tilt the apparatus and utilize a newspaper towel to remove the h2o).

4. Insert the comb, ensuring that at that place are no air bubbles.

5. Expect until the gel is solidified. (Tip: Solidification can be easily checked by leaving some gel solution in a tube).

Electrophoresis

1. Cascade the running buffer into the electrophorator [Figure 3].

   

   Add running buffer to the electrophorator

2. Place gel inside the electrophorator and connect to a power supply. (Tip: When connecting to the ability source always connect cerise to reddish, and black to black).

3. Brand certain buffer covers the gel completely, and remove the comb advisedly.

4. Load marking (half dozen μL) followed by samples (15 μL) in to each well [Figure 4].

   

   Add samples and molecular marker to the gel, afterwards removing the combs

5. Run the gel with low voltage (sixty V) for separating gel; utilise higher voltage (140 V) for stacking gel [Figure ​ 5a and ​ b].

   

   (a) Samples running through the stacking gel (lower voltage). (b): Samples running through the separating gel (college voltage)

6. Run the gel for approximately an hour, or until the dye front runs off the bottom of the gel [Figure 6].

   

   Run the gel to the lesser of the electrophorator

Electrotransfer

1. Cutting vi filter sheets to fit the measurement of the gel, and one polyvinylidene fluoride (PDVF) membrane with the same dimensions.

2. Wet the sponge and filter paper in transfer buffer, and wet the PDVF membrane in methanol.

3. Separate glass plates and recollect the gel.

4. Create a transfer sandwich every bit follows:

   Sponge

   3 Filter Papers

   Gel PVDF

   3 Filter Papers

   (Tip: Ensure there are no air bubbles betwixt the gel and PVDF membrane, and squeeze out extra liquid).

5. Relocate the sandwich to the transfer apparatus, which should be placed on ice to maintain 4°C. Add transfer buffer to the apparatus, and ensure that the sandwich is covered with the buffer. Place electrodes on height of the sandwich, ensuring that the PVDF membrane is between the gel and a positive electrode [Effigy 7].

   

   Transfer should exist done on water ice

6. Transfer for 90 minutes [Figure eight]. (Tip: The running fourth dimension should be proportional to the thickness of the gel, so this may exist reduced to 45 minutes for 0.75 mm gels).

   

Blocking and antibody incubation

1. Cake the membrane with five% skim milk in TBST^* for 1 hour.

2. Add chief antibody in 5% bovine serum albumin ( BSA) and incubate overnight in four°C on a shaker [Figure 9].

   

   Use a shaker to incubate the membrane with antibody

3. Wash the membrane with TBST for 5 minutes. Do this iii times. (Tip: All washing and antibiotic incubation steps should be washed on a shaker at room temperature to ensure fifty-fifty agitation).

4. Add secondary antibiotic in v% skim milk in TBST, and incubate for one hour.

5. Wash the membrane with TBST for v minutes. Do this 3 times

6. Prepare ECL mix (post-obit the proportion of solution A and B provided by the manufacturer). Incubate the membrane for 1–2 minutes [Figure ten]. (Tip: Use a 1000 μL pipette to ensure that ECL covers the top and bottom of the membrane).

   

   Incubate the membrane with ECL mix using a 1000 μL pipette to help the process

7. Visualize the result in the nighttime room [Effigy eleven]. (Tip: If the background is besides potent, reduce exposure time).

   

   Use the cassette to expose the membrane in the nighttime room

Recipe

1. Deliquesce the following in 800 ml of distilled H₂O

   • 8.eight g of NaCl

   • 0.2g of KCl

   • 3g of Tris base

2. Add 500ul of Tween-xx

3. Adjust the pH to seven.four

4. Add together distilled H₂O to 1L

5. Sterilize by filtration or autoclaving

Theory

Sample preparation

Cell lysates are the nigh common form of sample used for western blot. Protein extraction attempts to collect all the proteins in the cell cytosol. This should be done in a cold temperature with protease inhibitors to forbid denaturing of the proteins. Since tissue sample display a college caste of structure, mechanical invention, such every bit homogenization, or sonication is needed to excerpt the proteins.

After extracting the protein, it is very important to have a good idea of the extract's concentration. This eventually allows the researcher to ensure that the samples are existence compared on an equivalent basis. Poly peptide concentration is often measured using a spectrophotometer. Using this concentration allows to measure out the mass of the protein that is being loaded into each well by the relationship between concentration, mass, and volume.

After determining the appropriate volume of the sample, information technology is diluted into a loading buffer, which contains glycerol so that the samples sink hands into the wells of the gel. A tracking dye (bromophenol bluish) is also present in the buffer allowing the researcher to see how far the separation has progressed. The sample is heated after beingness diluted into a loading buffer, in social club to denature the higher order structure, while retaining sulfide bridges. Denaturing the high structure ensures that the negative charge of amino acids is not neutralized, enabling the protein to motion in an electrical field (applied during electrotransfer).

Information technology is likewise very important to have positive and negative controls for the sample. For a positive control a known source of target poly peptide, such equally purified poly peptide or a control lysate is used. This helps to confirm the identity 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 to confirm that the staining is not nonspecific.

Gel electrophoresis

Western blot uses 2 different types of agarose gel: stacking and separating gel. The college, stacking gel is slightly acidic (pH 6.8) and has a lower acrylamide concentration making a porous gel, which separates poly peptide poorly only allows them to class sparse, sharply defined bands. The lower gel, called the separating, or resolving gel, is basic (pH 8.8), and has a higher polyacrylamide content, making the gel's pores narrower. Protein is thus separated past their size more so in this gel, as the smaller proteins to travel more than hands, and hence chop-chop, than larger proteins.

The proteins when loaded on the gel have a negative accuse, as they take been denatured by heating, and will travel toward the positive electrode when a voltage is applied. Gels are unremarkably 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 and then connected to the power supply and allowed to run. The voltage is very of import, as a loftier voltage can overheat and distort the bands.

Blotting

After separating the poly peptide 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 betwixt the gel surface and the positive electrode in a sandwich. The sandwich includes a fiber pad (sponge) at each stop, 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 exist 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 exist done in semi-dry out or wet conditions. Wet weather are normally more reliable as it is less probable to dry the gel, and is preferred for larger proteins.

Associates of a sandwich in western Blot

The membrane, the solid back up, 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 retentiveness abilities. Notwithstanding, it is brittle, and does not allow the membrane to exist used for reprobing. In this regard, PVDF membranes provide better mechanical support and let the blot to be reprobed and stored. Yet, the background is higher in the PVDF membranes and therefore, washing advisedly is very important.

Washing, blocking and antibody incubation

Blocking is a very important stride of western blotting, every bit it prevents antibodies from binding to the membrane nonspecifically. Blocking is ofttimes made with 5% BSA or nonfat dried milk diluted in TBST to reduce the background.

Nonfat stale milk is often preferred every bit information technology is cheap and widely available. However, milk proteins are not compatible with all detection labels, then care must exist taken to choose the advisable blocking solution. For instance, BSA blocking solutions are preferred with biotin and AP antibody labels, and antiphosphoprotein antibodies, since milk contains casein, which is itself a phosphoprotein and biotin, thus interfering with the analysis results. It is often a expert strategy to incubate the primary antibody with BSA since information technology is ordinarily needed in higher amounts than the secondary antibiotic. Putting it in BSA solution allows the antibody to be reused, if the blot does non give good consequence.

The concentration of the antibody depends on the instruction by the manufacturer. The antibody tin be diluted in a wash buffer, such equally PBS or TBST. Washing is very important as information technology minimized background and removes unbound antibiotic. However, the membrane should not be left to launder for a really long fourth dimension, as it tin can also reduce the signal.

The membrane is then detected using the label antibody, usually with an enzyme such as horseradish peroxidase (HRP), which is detected by the signal it produces corresponding to the position of the target poly peptide. This bespeak is captured on a film which is usually developed in a dark room.

Quantification

It is very important to be aware that the data produced with a western blot is typically considered to exist semi-quantitative. This is because it provides a relative comparison of protein levels, just not an absolute measure of quantity. There are two reasons for this; first, in that location are variations in loading and transfer rates between the samples in separate lanes which are different on separate blots. These differences volition need to be standardized before a more precise comparison can be made. Second, the indicate generated past detection is non linear across the concentration range of samples. Thus, since the betoken produced is not linear, it should not be used to model the concentration.

Troubleshooting

Even though the procedure for western absorb is simple, many bug can arise, leading to unexpected results. The problem can be grouped into five categories: (i) unusual or unexpected bands, (2) no bands, (three) faint bands or weak signal, (4) loftier background on the absorb, and (five) patchy or uneven spots on the blot.

Unusual or unexpected bands can be due to protease degradation, which produces bands at unexpected positions. In this case information technology is advisable to use a fresh sample which had been kept on ice or alter the antibody. If the poly peptide seems to exist in too high of a position, then reheating the sample can help to intermission the quaternary protein structure. Similarly, blurry bands are often caused by high voltage or air bubbles present during transfer. In this example, it should be ensured that the gel is run at a lower voltage, and that the transfer sandwich is prepared properly. In add-on, changing the running buffer tin can also assist the problem. Nonflat bands tin be the effect of besides fast of a travel through the gel, due to low resistance. To prepare this the gel should be optimized to fit the sample. Finally, white (negative) bands on the picture are due to too much protein or antibody.

Another trouble: no bands tin can also arise due to many reasons related to antibody, antigen, or buffer used. If an improper antibiotic is used, either primary or secondary, the band will non show. In improver, the concentration of the antibody should exist appropriate equally well; if the concentration is too low, the signal may not exist visible. Information technology is important to recollect that some antibodies are not to be used for western absorb. Another reason for no visible bands is the lowest concentration or absence of the antigen. In this instance, antigen from another source tin be used to confirm whether the problem lies with the sample or with other elements, such as the antibiotic. Moreover, prolonged washing can also subtract the signal. Buffers can also contribute to the problem. It should exist ensured that buffers similar the transfer buffer, TBST, running buffer and ECL are all new and noncontaminated. If the buffers are contaminated with sodium azide, information technology can inactivate HRP.

Similarly, weak signals can be caused by low concentration of antibody or antigen. Increasing exposure time can also assistance to make the band clearer. Another reason could be nonfat dry milk masking the antigen. In this case use BSA or subtract the amount of milk used.

Loftier groundwork is oft caused past too high concentration of the antibody, which can bind to PVDF membranes. Another trouble could be the buffers, which may be likewise old. Increasing the washing time tin likewise aid to decrease the background. Additionally, besides high of an exposure tin too lead to this trouble. Therefore, information technology is advisable to check different exposure times to achieve an optimum time.

Patchy and uneven spots on the blot are normally caused past improper transfer. If there are air bubbling trapped betwixt the gel and the membrane, information technology volition announced darker on the motion picture. It is besides of import to apply a shaker for all incubation, so that there is no uneven agitation during the incubation. In one case once again, washing is of utmost importance also to launder the groundwork. This problem can too be caused by antibodies bounden to the blocking agents; in this case another blocking agent should exist tried. Filtering the blocking agent tin also assist to remove some contaminants. Finally, this trouble can besides be acquired past aggregation of the secondary antibiotic; in this example, the secondary antibody should exist centrifuged and filtered to remove the aggregated.

Decision

Western blot is a technique that is very useful for protein detection as it allows the user to quantify the protein expression equally well. This paper covered the protocol, the theory behind that protocol, and some troubleshooting techniques. Western blot can be seen as an intricate remainder, every bit the researcher attempts to get a nonspecific, nevertheless strong point.

Footnotes

Source of Support: Aught.

Disharmonize of Interest: None declared.

^*TBST: Tris-Buffered Saline Tween-20

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/