Western blot transfer issues

Western blot transfer issues

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I am having some issues with transfer efficiency to nitrocellulose membranes. To get all the technical info out of the way:

Transfer method: Wet, tank transfer overnight at 30V with normal tris-glycine transfer buffer + 20% methanol. I do this at RT with stirring. Of note: I have not recently been equilibrating my gel in transfer buffer, as this was reported to decrease transfer of basic proteins due to the stripping of SDS (I can furnish this reference if anyone is interested).

Sample: In all cases these are in vitro reconstituted nucleosomes with or without certain enzymes--the main transfer issues are the core histones, which are highly basic (pI 10-11). There should be no more than about 100 ng of each histone being transferred per lane. The salt in the sample is <25 mM. They are boiled prior to loading. There is no noticeable precipitate in the samples before or after boiling, however the volume is quite small (20 ul) so I would not necessarily detect precipitation.

Nitrocellulose is 0.2 um and is pre-equilibrated in transfer buffer prior to transfer.

The gel: I use a 15% gel, 1.0-1.5 mm thick. The acrylamide I use is 37.5:1 acrylamide:bis. Some issues could arise here--the acrylamide solution is about 1 year old and has been stored at RT that entire time (which is what is recommended on the label). In addition, we typically keep a stock of 10% APS at 4C, so I cannot guarantee my APS has not gone bad.

I have been quite careful generally to ensure there are no bubbles between any components of the sandwich and filter paper/foam pads are soaked in transfer buffer.

Now to the issue. Attached are images of the membrane after transfer (and also after processing, thus the relatively high background from the blocking buffer) as well as the gel after transfer. As you can see from lane to lane I seem to have differing transfer efficiencies. This has occurred with my last three blots. My first thought was sample precipitation, but I have not had this issue before despite running essentially the same blot many many times over the past year.

Any thoughts? Quite frustrating, as it has appeared suddenly with identical protocols/reagents as before.

The first thing that comes to mind for me is that the transfer seems excessive… isn't histone sub-20 kDa? I have often transferred similar-sized proteins at 4C for 30 min, 30V.

In general, presence of SDS will inhibit transfer efficiency, and increasing alcohol in your TG buffer will counteract the effect of SDS present. Perhaps bring down the methanol to 10%? I would be curious to see that reference regarding the SDS/pre-equilibration, though. That certainly runs contrary to how I understand it working. (Note: I flip-flop between equilibrating or not equilibrated my gels without any observable differences.)

If your acrylamide or ACS had gone bad, your gel wouldn't set.

If you don't already, I'd suggest running a Bradford or BCA on your samples to be sure that you're loading the same amount into each well. Having loaded more into each well could give the appearance of uneven transfer.

To be honest, I don't really see what you mean by uneven transfer, based off the Coomassie and Ponceau. If you're seeing a gradient effect across a large portion of the gel, I would suggest you visually inspect to make sure that the gels you cast are even width all the way across. Having a thicker gel in one place than in another could impact the resistance (heat) in that part of the gel and impact transfer. If you're seeing a lane-by-lane transfer efficiency shift, I would suggest localized pH or temperature changes due to uneven loading of salts, protein concentration, or lipid concentration.

Hope this helps!

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.

Conflict of interest statement

Conflict of Interest: None declared.


Assembled rack for gel solidification

Assembled rack for gel solidification

Add gel solution using a transfer pipette

Add running buffer to the…

Add running buffer to the electrophorator

Add samples and molecular marker…

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

(a) Samples running through the…

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


Western blot is usually utilized in analysis to separate and establish proteins. In this system a mix of proteins is separated primarily based on molecular weight, and thus by kind, by means of gel electrophoresis. These outcomes are then transferred to a membrane producing a band for every protein. The membrane is then incubated with labels antibodies particular to the protein of curiosity.

The unbound antibody is washed off leaving solely the certain antibody to the protein of curiosity. The certain antibodies are then detected by growing the movie. As the antibodies solely bind to the protein of curiosity, just one band needs to be seen. The thickness of the band corresponds to the quantity of protein current thus doing an ordinary can point out the quantity of protein current. The paper will first describe the protocol for western blot, accompanied by footage to assist the reader and idea to rationalize the protocol. This can be adopted by the theoretical clarification of the process, and within the later part, troubleshooting suggestions for widespread issues.


Cell lysis to extract protein

Protein might be extracted from totally different form of samples, reminiscent of tissue or cells. Below is the protocol to extract proteins from adherent cells.

  1. Wash cells within the tissue tradition flask or dish by including chilly phosphate buffered saline (PBS) and rocking gently. Discard PBS. (Tip: Keep tissue tradition dish on ice all through).
  2. Add PBS and use a cell scraper to dislodge the cells. Pipette the combination into microcentrifuge tubes.
  3. Centrifuge at 1500 RPM for five minutes and discard the supernatant.
  4. Add 180 μL of ice chilly cell lysis buffer with 20 μL contemporary protease inhibitor cocktail. (Tip: If protein focus just isn’t excessive sufficient on the finish, it’s suggested to repeat the process with a better proportion of protease inhibitor cocktail).
  5. Incubate for 30 minutes on ice, and then make clear the lysate by spinning for 10 minutes at 12,000 RPM, at 4°C.
  6. Transfer supernatant (or protein combine) to a contemporary tube and retailer on ice or frozen at -20°C or -80°C.
  7. Measure the focus of protein utilizing a spectrophotometer.

Sample preparation

  1. decide the amount of protein extract to make sure 50 μg in every effectively.
  2. Add 5 μL pattern buffer to the pattern, and make the amount in every lane equalized utilizing double distilled H2O (dd H2O). Mix effectively. (Tip: Total quantity of 15 μL per lane is usually recommended).
  3. Heat the samples with dry plate for five minutes at 100°C.

Gel preparation

  1. After making ready the 10% stacking gel answer, assemble the rack for gel solidification [Figure 1]. (Tip: 10% AP and TEMED solidify the answer due to this fact, each gels might be ready on the identical time, if the abovementioned reagents usually are not added till the top).Figure 1Assembled rack for gel solidification
  2. Add stacking gel answer fastidiously till the extent is the same as the inexperienced bar holding the glass plates [Figure 2]. Add H2O to the highest. Wait for 15–30 minutes till the gel turning solidified. (Tip: Using a suction pipette could make the method of including the gel to the glass plate simpler).Open in a separate windowFigure 2Add gel answer utilizing a switch pipette
  3. Overlay the stacking gel with the separating gel, after eradicating the water. (Tip: It is healthier to tilt the equipment and use a paper towel to take away the water).
  4. Insert the comb, making certain that there aren’t any air bubbles.
  5. Wait till the gel is solidified. (Tip: Solidification might be simply checked by leaving some gel answer in a tube).


  1. Pour the working buffer into the electrophorator [Figure 3].Open in a separate windowFigure 3Add working buffer to the electrophorator
  2. Place gel contained in the electrophorator and connect with an influence provide. (Tip: When connecting to the ability supply all the time join crimson to crimson, and black to black).
  3. Make positive buffer covers the gel utterly, and take away the comb fastidiously.
  4. Load marker (6 μL) adopted by samples (15 μL) in to every effectively [Figure 4].Open in a separate windowFigure 4Add samples and molecular marker to the gel, after eradicating the combs
  5. Run the gel with low voltage (60 V) for separating gel use greater voltage (140 V) for stacking gel [Figure ​[Figure5a5a and ​andbb].Figure 5(a) Samples working by means of the stacking gel (decrease voltage). (b): Samples working by means of the separating gel (greater voltage)
  6. Run the gel for about an hour, or till the dye entrance runs off the underside of the gel [Figure 6].Figure 6Run the gel to the underside of the electrophorator


  1. Cut 6 filter sheets to suit the measurement of the gel, and one polyvinylidene fluoride (PDVF) membrane with the identical dimensions.
  2. Wet the sponge and filter paper in switch buffer, and moist the PDVF membrane in methanol.
  3. Separate glass plates and retrieve the gel.
  4. Create a switch sandwich as follows:Sponge3 Filter PapersGel PVDF3 Filter Papers(Tip: Ensure there aren’t any air bubbles between the gel and PVDF membrane, and squeeze out further liquid).
  5. Relocate the sandwich to the switch equipment, which needs to be positioned on ice to take care of 4°C. Add switch buffer to the equipment, and make sure that the sandwich is roofed with the buffer. Place electrodes on high of the sandwich, making certain that the PVDF membrane is between the gel and a optimistic electrode [Figure 7].Figure 7Transfer needs to be executed on ice
  6. Transfer for 90 minutes [Figure 8]. (Tip: The working time needs to be proportional to the thickness of the gel, so this can be diminished to 45 minutes for 0.75 mm gels).Open in a separate windowFigure 8Membrane after switch

Blocking and antibody incubation

  1. Block the membrane with 5% skim milk in TBST * for 1 hour.
  2. Add major antibody in 5% bovine serum albumin ( BSA) and incubate in a single day in 4°C on a shaker [Figure 9].Figure 9Use a shaker to incubate the membrane with antibody
  3. Wash the membrane with TBST for five minutes. Do this Three occasions. (Tip: All washing and antibody incubation steps needs to be executed on a shaker at room temperature to make sure even agitation).
  4. Add secondary antibody in 5% skim milk in TBST, and incubate for 1 hour.
  5. Wash the membrane with TBST for five minutes. Do this Three occasions
  6. Prepare ECL combine (following the proportion of answer A and B supplied by the producer). Incubate the membrane for 1–2 minutes [Figure 10]. (Tip: Use a 1000 μL pipette to make sure that ECL covers the highest and backside of the membrane).Figure 10Incubate the membrane with ECL combine utilizing a 1000 μL pipette to assist the method
  7. Visualize the end result in the dead of night room [Figure 11]. (Tip: If the background is simply too robust, cut back publicity time).Figure 11Use the cassette to reveal the membrane in the dead of night room


  1. Dissolve the next in 800 ml of distilled H2O
    • 8.Eight g of NaCl
    • 0.2g of KCl
    • 3g of Tris base
  2. Add 500ul of Tween-20
  3. Adjust the pH to 7.4
  4. Add distilled H2O to 1L
  5. Sterilize by filtration or autoclaving


Sample preparation

Cell lysates are the commonest type of pattern used for western blot. Protein extraction makes an attempt to gather all of the proteins within the cell cytosol. This needs to be executed in a chilly temperature with protease inhibitors to stop denaturing of the proteins. Since tissue pattern show a better diploma of construction, mechanical invention, reminiscent of homogenization, or sonication is required to extract the proteins.

After extracting the protein, it is extremely essential to have a good suggestion of the extract’s focus. This ultimately permits the researcher to make sure that the samples are being in contrast on an equal foundation. Protein focus is usually measured utilizing a spectrophotometer. Using this focus permits to measure the mass of the protein that’s being loaded into every effectively by the connection between focus, mass, and quantity.

After figuring out the suitable quantity of the pattern, it’s diluted right into a loading buffer, which comprises glycerol in order that the samples sink simply into the wells of the gel. A monitoring dye (bromophenol blue) can also be current within the buffer permitting the researcher to see how far the separation has progressed. The pattern is heated after being diluted right into a loading buffer, so as to denature the upper order construction, whereas retaining sulfide bridges. Denaturing the excessive construction ensures that the unfavorable cost of amino acids just isn’t neutralized, enabling the protein to maneuver in an electrical area (utilized throughout electrotransfer).

It can also be crucial to have optimistic and unfavorable controls for the pattern. For a optimistic management a identified supply of goal protein, reminiscent of purified protein or a management lysate is used. This helps to verify the identification of the protein, and the exercise of the antibody. A unfavorable management is a null cell line, reminiscent of β-actin, is used as effectively to verify that the staining just isn’t nonspecific.

Gel electrophoresis

Western blot makes use of two various kinds of agarose gel: stacking and separating gel. The greater, stacking gel is barely acidic (pH 6.8) and has a decrease acrylamide focus making a porous gel, which separates protein poorly however permits them to kind skinny, sharply outlined bands. The decrease gel, referred to as the separating, or resolving gel, is primary (pH 8.8), and has a better polyacrylamide content material, making the gel’s pores narrower. Protein is thus separated by their measurement extra so on this gel, because the smaller proteins to journey extra simply, and therefore quickly, than bigger proteins.

The proteins when loaded on the gel have a unfavorable cost, as they’ve been denatured by heating, and will journey towards the optimistic electrode when a voltage is utilized. Gels are often made by pouring them between two glass or plastic plates, utilizing the answer described within the protocol part. The samples and a marker are loaded into the wells, and the empty wells are loaded with pattern buffer. The gel is then related to the ability provide and allowed to run. The voltage is essential, as a excessive voltage can overheat and distort the bands.


After separating the protein combination, it’s transferred to a membrane. The switch is completed utilizing an electrical area oriented perpendicular to the floor of the gel, inflicting proteins to maneuver out of the gel and onto the membrane. The membrane is positioned between the gel floor and the optimistic electrode in a sandwich. The sandwich features a fiber pad (sponge) at every finish, and filter papers to guard the gel and blotting membrane [Figure 12]. Here two issues are crucial: (1) the shut contact of gel and membrane to make sure a transparent picture and (2) the position of the membrane between the gel and the optimistic electrode. The membrane should be positioned as such, in order that the negatively charged proteins can migrate from the gel to the membrane. This kind of switch is named electrophoretic switch, and might be executed in semi-dry or moist situations. Wet situations are often extra dependable as it’s much less more likely to dry out the gel, and is most popular for bigger proteins.Figure 12

Assembly of a sandwich in western Blot

The membrane, the strong assist, is a vital a part of this course of. There are two sorts of membrane: nitrocellulose and PVDF. Nitrocellulose is used for its excessive affinity for protein and its retention skills. However, it’s brittle, and doesn’t enable the membrane for use for reprobing. In this regard, PVDF membranes present higher mechanical assist and enable the blot to be reprobed and saved. However, the background is greater within the PVDF membranes and due to this fact, washing fastidiously is essential.

Washing, blocking and antibody incubation

Blocking is a vital step of western blotting, because it prevents antibodies from binding to the membrane nonspecifically. Blocking is usually made with 5% BSA or nonfat dried milk diluted in TBST to scale back the background.

Nonfat dried milk is usually most popular as it’s cheap and extensively obtainable. However, milk proteins usually are not suitable with all detection labels, so care should be taken to decide on the suitable blocking answer. For instance, BSA blocking options are most popular with biotin and AP antibody labels, and antiphosphoprotein antibodies, since milk comprises casein, which is itself a phosphoprotein and biotin, thus interfering with the assay outcomes. It is usually a very good technique to incubate the first antibody with BSA since it’s often wanted in greater quantities than the secondary antibody. Putting it in BSA answer permits the antibody to be reused, if the blot doesn’t give good end result.

The focus of the antibody relies on the instruction by the producer. The antibody might be diluted in a wash buffer, reminiscent of PBS or TBST. Washing is essential because it minimized background and removes unbound antibody. However, the membrane shouldn’t be left to clean for a very very long time, as it may well additionally cut back the sign.

The membrane is then detected utilizing the label antibody, often with an enzyme reminiscent of horseradish peroxidase (HRP), which is detected by the sign it produces similar to the place of the goal protein. This sign is captured on a movie which is often developed in a darkish room.


It is essential to remember that the info produced with a western blot is often thought of to be semi-quantitative. This is as a result of it supplies a relative comparability of protein ranges, however not an absolute measure of amount. There are two causes for this first, there are variations in loading and switch charges between the samples in separate lanes that are totally different on separate blots. These variations will must be standardized earlier than a extra exact comparability might be made. Second, the sign generated by detection just isn’t linear throughout the focus vary of samples. Thus, because the sign produced just isn’t linear, it shouldn’t be used to mannequin the focus.


Even although the process for western blot is straightforward, many issues can come up, resulting in sudden outcomes. The drawback might be grouped into 5 classes: (1) uncommon or sudden bands, (2) no bands, (3) faint bands or weak sign, (4) excessive background on the blot, and (5) patchy or uneven spots on the blot.

Unusual or sudden bands might be resulting from protease degradation, which produces bands at sudden positions. In this case it’s advisable to make use of a contemporary pattern which had been stored on ice or alter the antibody. If the protein appears to be in too excessive of a place, then reheating the pattern might help to interrupt the quaternary protein construction. Similarly, blurry bands are sometimes attributable to excessive voltage or air bubbles current throughout switch. In this case, it needs to be ensured that the gel is run at a decrease voltage, and that the switch sandwich is ready correctly. In addition, altering the working buffer may also assist the issue. Nonflat bands might be the results of too quick of a journey by means of the gel, resulting from low resistance. To repair this the gel needs to be optimized to suit the pattern. Finally, white (unfavorable) bands on the movie are resulting from an excessive amount of protein or antibody.

Another drawback: no bands may also come up resulting from many causes associated to antibody, antigen, or buffer used. If an improper antibody is used, both major or secondary, the band is not going to present. In addition, the focus of the antibody needs to be acceptable as effectively if the focus is simply too low, the sign will not be seen. It is essential to keep in mind that some antibodies usually are not for use for western blot. Another purpose for no seen bands is the bottom focus or absence of the antigen. In this case, antigen from one other supply can be utilized to verify whether or not the issue lies with the pattern or with different parts, such because the antibody. Moreover, extended washing may also lower the sign. Buffers may also contribute to the issue. It needs to be ensured that buffers just like the switch buffer, TBST, working buffer and ECL are all new and noncontaminated. If the buffers are contaminated with sodium azide, it may well inactivate HRP.

Similarly, weak indicators might be attributable to low focus of antibody or antigen. Increasing publicity time may also assist to make the band clearer. Another purpose might be nonfat dry milk masking the antigen. In this case use BSA or lower the quantity of milk used.

High background is usually attributable to too excessive focus of the antibody, which might bind to PVDF membranes. Another drawback might be the buffers, which can be too previous. Increasing the washing time may also assist to lower the background. Additionally, too excessive of an publicity may also result in this drawback. Therefore, it’s advisable to verify totally different publicity occasions to attain an optimum time.

Patchy and uneven spots on the blot are often attributable to improper switch. If there are air bubbles trapped between the gel and the membrane, it’ll seem darker on the movie. It can also be essential to make use of a shaker for all incubation, in order that there is no such thing as a uneven agitation through the incubation. Once once more, washing is of utmost significance as effectively to clean the background. This drawback may also be attributable to antibodies binding to the blocking brokers on this case one other blocking agent needs to be tried. Filtering the blocking agent may also assist to take away some contaminants. Finally, this drawback may also be attributable to aggregation of the secondary antibody on this case, the secondary antibody needs to be centrifuged and filtered to take away the aggregated.Go to:

Transfer Buffers

Transfer buffers contain a conductive, strong buffering agent (for example, Tris, CAPS, or carbonate) in order to maintain the conductivity and pH of the system during transfer. In addition, alcohol (methanol or ethanol) may be included in the transfer buffer to promote binding of proteins to membranes, and SDS may be added to promote elution of proteins from gels.

Compatible Transfer Buffer by Gel Type

Gel Type Transfer Buffer Western Blotting Transfer System
SDS-PAGE Towbin with or without SDS, CAPS, carbonate, Bjerrum Schafer-Nielsen Tank blotting or semi-dry blotting
Tris-Tricine Towbin, CAPS Tank blotting recommended needs high-capacity, small pore-size membrane pH of buffer may be critical
Native, non-denaturing Depends on pH of gel buffer and pI of protein of interest Tank blotting recommended temperature regulation may be needed to maintain activity
Acid urea 0.7% acetic acid Use acid-gel transfer protocol (membrane toward cathode)

SDS and Alcohol

SDS and alcohol play opposing roles in a transfer. SDS promotes elution of the protein from the gel and in cases where certain proteins are difficult to elute from the gel, SDS may be added to the transfer buffer. However, SDS in the transfer buffer decreases the binding efficiency of protein to nitrocellulose membrane PVDF membrane can be substituted if desired.

Alcohol, on the other hand, removes the SDS from SDS-protein complexes and improves the binding of protein to nitrocellulose membrane. But alcohol may cause a reduction in the gel pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral.

Recommended Transfer Buffer by Application

Application Recommended Transfer Buffer
High-molecular-weight proteins Towbin with SDS
Small proteins and peptides Towbin, CAPS
Basic proteins (pI > 9) in denaturing gels CAPS, carbonate, Bjerrum Schafer-Nielsen
Basic proteins (pI > 9) in native or non-denaturing gels 0.7% acetic acid
Glycoproteins Towbin with or without SDS, CAPS, carbonate, Bjerrum Schafer-Nielsen nondenaturing gels
Proteoglycans Towbin, Bjerrum Schafer-Nielsen

Transfer Buffer Tips

  • Do not use the same batch of transfer buffer more than once, as the buffer will likely lose its capacity to maintain a stable pH during transfer
  • Do not dilute transfer buffers this will also decrease buffering capacity
  • Do not adjust the pH of transfer buffers when not indicated, as this increases buffer conductivity, which is manifested by higher initial current output and decreased resistance

Constitutive Activity in Receptors and Other Proteins, Part B

Mercedes Dosil , James B. Konopka , in Methods in Enzymology , 2010

7.2 Western blot analysis of receptor protein production

Western blot analysis is another very convenient method to detect Ste2 proteins produced in yeast. We use a triple HA tag, which provides a very sensitive level of detection ( Dosil et al., 2000 ). The preparation of yeast cell extracts requires disruption of the cell wall. Mechanical disruption by vortexing cells with glass beads is the simplest and will be described here. Whole cell extracts can be analyzed, but sensitivity can be increased by analyzing a crude membrane fraction. Methods to separate the protein extract by polyacrylamide gel electrophoresis and transfer to nitrocellulose are common practices that are described in detail elsewhere ( Sambrook et al., 1989 ).

Acid treated 450 μm glass beads (e.g., Sigma cat.# G8772).

TE/PP buffer: 100 mM NaCl, 50 mM Tris–HCl (pH 7.5), 1 mM EDTA, 100 μg/ml PMSF, 2 μg/ml pepstatin A.

PAGE sample buffer (8 M urea, 3% SDS, 25 mM Tris (pH 6.8), 0.01% bromophenol blue, 0.01% xylene cyanol).

Grow a 50 mL culture of yeast to a density of about 1 × 10 7 cells/ml (OD660 = 0.5–1). Either YPD or synthetic medium can be used.

Cool centrifuge tubes and buffers on ice.

Transfer about 2.5 × 10 8 cells to a 50 ml conical tube and harvest cell pellet by centrifugation at 1000×g for 5 min.

Wash cell pellet with 10 mL sterile water and then pellet cells again by centrifugation for 5 min.

Resuspend cell pellet in 1 mL sterile water and transfer to 1.5 mL microfuge tube.

Centrifuge at 14,000×g for 30 s in a microfuge and then remove the supernatant. (Cells may be stored at − 70 °C at this point or placed on ice until needed.)

Resuspend 2.5 × 10 8 cells in 250 μL TE/PP buffer and add 250 μL glass beads.

Vortex cells at very high speed for 1 min. Tilt tube so that there is maximum smashing action of the glass beads to break the yeast cell walls. Incubate on ice for 1 min to keep the sample cool and prevent proteolysis. Repeat three times.

Transfer cell extract to fresh tube (leaving behind the glass beads).

Centrifuge at low speed (330×g) for 5 min at 4 °C to remove unbroken cells.

Transfer supernatant to a new tube. Centrifuge at top speed for 15 min at 4 °C in a microfuge to pellet membranes.

The crude membrane pellet can be used immediately or stored at − 70 °C.

Dissolve the crude membrane pellet in 100 μL 2× PAGE loading buffer. Warm tubes at 37 °C for 10 min prior to loading gel. Do not boil GPCR samples or they will aggregate.

Centrifuge at top speed in a microfuge for 3 min prior to loading and then perform gel electrophoresis and Western blot according to standard procedures.

Step 5: Detection

The detection method used is dependent upon the enzyme to which the secondary antibody is conjugated. The most common enzyme used in Western Blotting is HRP, and the substrate used for detection is known as chemiluminescent substrate.

Once the substrate has been added, the light being emitted can be detected with film or a photo imager.

Notice that the film does not contain information on where the lit bands are located in relation to the membrane. Therefore, it is very important to use a method that allows the film to be aligned with the membrane in the exact same manner as when the film was exposed. The membrane is usually stained after the detection step, so that the protein present can be visualized, and compared to the film.

Chemiluminescent Detection

Follow manufacturer’s instructions.

  1. Prepare the substrate according to manufacturer’s instructions.
  2. Place the blot in a container and add substrate to completely cover the membrane.
    Incubate for 1 minute.
  3. Drain excess substrate.
  4. Place the blot on a clean piece of glass and wrap in plastic wrap.
    Note: A cut-to-size sheet protector or a freezer bag can also be used.
  5. Gently smooth out any air bubbles.
  6. In a dark room, place the wrapped membrane in a film cassette.
  7. Place a sheet of autoradiography film on top and close the cassette.
  8. Expose film. Multiple exposures of 15 seconds to 30 minutes should be done to determine the optimum exposure time 1 to 5 minutes is common.

Western Blot Doctor™ — Blot Background Problems


The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. In this section, you can find solutions to problems with blot background signal.

Other sections in the Western Blot Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem.

Problem: White spots or regions on blot

Foam pads for Bio-Rad wet tank blotting systems:

  • Mini Trans-Blot ® System (1703933)
  • Criterion&trade Blotter (1704086)
  • Trans-Blot System (1703914)
  • Trans-Blot Plus System (1703995)

As a diagnostic test, you can check transfer quality by imaging proteins on the gel and blot. If planning to use the blot in downstream steps, make sure that your stain can be removed or is compatible with antibody detection. For example, Coomassie and colloidal gold are not compatible with downstream steps (see Bio-Rad Protein Stains and the Protein Stain Selection Guide).

  • High-intensity transfers in tank blotting sample can cause buffer heating, leading to bubble formation ensure that buffer remains cool
    • Reduce current, and increase transfer time to compensate
    • Perform transfer in tank apparatus placed in ice or a temperature-controlled (4°C) room
    • Chill buffers before use
    • Use a cooling coil or &ldquoblue ice&rdquo insert in the cell
    • Mini Trans-Blot System (1703919)
    • Criterion Blotter (1704076, 1704087)
    • Trans-Blot System (1703912)
    • Trans-Blot Plus System (1703990)
    • Nitrocellulose: White regions on the nitrocellulose membrane indicate dry areas where protein will not bind. If wetting does not occur immediately by immersion of the sheet in transfer buffer, heat distilled water until just under boiling point and soak the membrane until completely wet. Equilibrate in transfer buffer until ready to use
    • PVDF: White regions on PVDF membrane indicate areas where the membrane was either inappropriately prewetted or allowed to dry out. Because of the hydrophobic nature of PVDF, the membrane must be prewetted in methanol prior to equilibration in aqueous transfer buffer. Once wet, do not allow membrane to dry out. If the membrane dries, rewet in methanol and re-equilibrate in TBS-T (caution: may adversely affect downstream detection processes)

    Problem: High background on blot

    • Increase the concentration of blocker (e.g., 3&ndash5% BSA, casein, or nonfat dry milk)
    • Increase the duration of the blocking step (overnight at 4°C instead of 1 hour at room temperature, or a longer incubation at room temperature)
    • Increase temperature at which blocking is performed, (up to room temp)
    • Use a different blocking reagent (albumin, gelatin, BSA, casein, or nonfat dry milk)
    • More blocking reagents:
    • Use a pure protein such as BSA or casein as a blocker (see Detergents and Blocking Reagents)
    • Do not reuse blocking buffers
    • Reduce incubation time with detection substrate
    • If using colorimetric reagent, remove the blot from the substrate solution when the signal-to-noise level is acceptable, and immerse in deionized water
    • Reduce/optimize primary and/or secondary antibody concentrations
    • Use a dot-blotting trial to optimize antibody concentrations
    • Reduce/optimize incubation times
    • Make sure all incubation trays are fully cleaned between experiments
    • Use disposable trays
    • Excessive protein loading
    • Too much SDS in transfer buffer
    • Excess protein loading:
    • Reduce the amount of protein on gel
    • Optimize sample loading see Determining the Appropriate Sample Load for Western Blots Protocol
    • Reduce concentration of SDS in transfer buffer
    • Add second membrane sheet to bind excess protein
    • Try other blocking reagents, e.g., albumin, gelatin, BSA, casein, or nonfat dry milk (see Bio-Rad Detergents and Blocking Reagents and more Blocking Reagents)
    • Do not use milk to block membranes when using avidin-biotin system because milk contains biotin
    • Increase length and/or number of washing steps (minimum of 5 x 5 min)
    • Use larger volume of wash buffer
    • Use a shorter exposure time
    • Use multi-acquisition feature on data acquisition software
    • Film users:
      • Wait 5&ndash10 minutes and then re-expose blot to film (film)
      • Reduce exposure and/or development time (film)
      • Consider switching to a digital imaging system such as Bio-Rad&rsquos ChemiDoc&trade Imaging Systems
      • Make sure membrane is thoroughly wetted when beginning procedure
      • Repeat procedure taking care that the blot does not dry out during any step by using sufficient volumes and agitation throughout
      • Make sure membrane remains submerged in incubation and wash buffers throughout all steps
      • Use a fresh aliquot of antibody that has been stored at &ndash20°C or below
      • If storing an antibody for a very long period of time may want to store at &ndash80°C
      • Make aliquots of antibody and only thaw one at a time as needed for blots
      • Avoid repeated freeze-thaw cycles
      • Try a lower incubation temperature such as 4°C
        Note: will need to increase incubation time
      • Try nitrocellulose instead (see Western Blotting Membranes)
      • Increase stringency of washing steps:
      • Use TBS containing 0.05&ndash0.1% Tween 20
      • Try a stronger detergent such as NP-40
      • Use fresh buffers
      • Filter all buffers through a 0.2 µm filter before using for washing or blocking and before adding antibody

      Problem: Blotchy or patchy background

      • Use a shaker during all incubation steps
      • Make sure membrane is thoroughly wetted when beginning procedure
      • Repeat procedure taking care that the blot does not dry out during any step by using sufficient volumes and agitation throughout. Make sure blot is always submerged
      • Do not handle membrane with bare hands. Always wear clean gloves, and handle blots with clean forceps whenever possible
      • Make sure electrophoresis equipment, blotting equipment, and incubation trays are clean and free of contaminants
      • Avoid touching the blot to surfaces
      • Increase washing buffer volume
      • Use a shaker during all incubation and wash steps
      • Avoid stacking blots in a single incubation container
        • When stacking blots in a single incubation container, ensure that sufficient flow of buffer exists between blots. The blots should move freely past each other with agitation during incubation and wash steps

        Problem: Uneven spots on blot or speckled background

        • Spin secondary antibody and/or filter to remove aggregates
        • Make sure blocking reagent is fully dissolved in buffer prior to use
        • Make fresh buffers if making blocking buffer from dry reagents
        • Check premade blocking buffers for precipitates before use
        • Add 0.05&ndash0.1% Tween 20 to blocking buffer
        • Filter blocking buffer through 0.2 µm filter before use
        • Wash blot with washing buffer before incubation with antibodies
        • Try a different blocking reagent, e.g., albumin, gelatin, BSA, casein, or nonfat dry milk (see Bio-Rad Detergents and Blocking Reagents and more Blocking Reagents)
        • Remove all residual acrylamide gel traces from surface of membrane following transfer before proceeding with downstream steps
        • Check that scanning and cassette surfaces are clean
        • Use fresh buffers. Make a new batch of buffer and repeat blot
        • Filter buffers for blocking and washing through 0.2 µm filter
        • Wrapping blots in plastic wrap or heat-sealed bags is a good safeguard against both particulate contamination and blot dehydration at the image acquisition step

        Problem: Gel cassette shadow transferred to blot (tank blotters)

        • Clean or replace foam pads
          • Foam pads for Bio-Rad wet tank blotting systems:
            • Mini Trans-Blot ® System (1703933)
            • Criterion&trade Blotter (1704086)
            • Trans-Blot System (1703914)
            • Trans-Blot Plus System (1703995)
            • Reduce amount of protein on the gel
            • Optimize sample loading see Determining the Appropriate Sample Load for Western Blots Protocol
            • Reduce the amount of SDS in transfer buffer
            • Add second membrane sheet to bind excess protein
            • Prepare fresh transfer buffer. Make a new batch of buffer and repeat blot
            • Filter buffers for blocking and washing through 0.2 µm filter


            Watch the video: SDS-PAGE + Wet transfer Western blot (July 2022).


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