Azure BiosystemsHigh-quality and affordable imaging systems, qPCR systems, and reagents.
Azure BiosystemsAt Azure Biosystems, we develop easy-to-use, high-performance imaging systems and high-quality reagents optimized for generating quantitative data from western blots. From our innovative, first-in-class hybrid laser scanner-imager, the Sapphire Biomolecular Imager, to our Radiance-Q ECL substrate which has an exceptionally wide dynamic range and long half-life, we provide performance in places other companies simply overlook. Here are just a few of these excellent products. See our full portfolio of western blotting instruments and reagents by visiting azurebiosystems.com/.
Our workhorse western blot and gel imager for detecting RGB fluorescence, NIR fluorescence, and chemiluminescence.
Azure Imagers are available in a range of configurations to support your needs now, and are upgradable to support your future needs.
RGB | NIR | Chemi | Blue/Color/UV | |
|---|---|---|---|---|
600 | ✓ | ✓ | ✓ | ✓ |
500 | ✓ | ✓ | ✓ | |
400 | ✓ | ✓ | ✓ | |
300 | ✓ | ✓ | ||
280 | ✓ | ✓ | ||
200 | ✓ |
Our hybrid laser scanner-imagers for sensitive, high-resolution western blot and gel imaging plus a whole lot more.
Image in-cell westerns, 2D-gels, and numerous other applications using RGB fluorescence, NIR fluorescence, chemiluminescence, and phosphor imaging.
Multiple configurations available to maximize your flexibility.
Like all assays, the quality and quantitativeness of western blot data is only as good as the weakest, most variable link in the measurement process. No matter the quality, accuracy, and precision of everything else, the step where accuracy and reproducibility are the poorest will be the step that defines and limits how quantitative your western blot will be. Which is why we’ve created this guide. In these pages, we provide practical, hands-on recommendations on how to obtain high-quality, quantitative western blot data so that you can draw solid conclusions and generate insights you trust.
Before discussing how to get quantitative data from a western blot, it’s useful to define what we mean by “quantitative.” For a measurement to be quantitative, it must meet the following criteria:
Laying out the definition of “quantitative” in this way provides a guidepost for the steps we can take to ensure that western blot data is quantitative—the western blot process needs to be well-defined and the steps followed as consistently as possible, the data need to be reproducible, and the data should be consistent with data collected using an orthogonal method such as mass spectrometry.
In the rest of this section we focus on how to meet these criteria, highlighting where paying special attention to consistency or performing reagent validations and controls can make a measurable impact on the reliability and reproducibility of your western blot data.
“Just because we can put numbers on an image does not imply that we should—a quantified biomolecule should relate directly to the true quantity of that biomolecule if it is to be meaningful.”1
QUANTITATIVE VS. QUALITATIVE
A qualitative Western blot shows the presence or absence of a protein of interest but provides no information on relative protein abundance. A quantitative Western blot is used to detect specific proteins and measure relative protein abundance.
PRECISION
How close measurements are to each other
ACCURACY
How close a measurement is to the “true” measurement
Consistent sample preparation and handling is an important first step for generating quantitative westerns. Even small differences in incubation times, buffer composition, or temperatures (variable room temperature, freezing at -20°C vs. holding on ice), can noticeably detract from the reproducibility of your western blot data2. Once you establish a reliable protocol for cell or tissue lysis and sample handling, follow the procedure as closely as possible when preparing samples that will be compared to each other.
“Quantitation of Western blots is not always required but it can be fraught with traps for the unwary investigator and often sparks lively debate among scientists.”4
Journals want to see that an antibody does, in fact, detect the protein of interest, and it’s just good experimental technique to validate antibodies in your own lab using your own samples and techniques. This is especially important when using an antibody that you have never published with before.
Simply verifying that your primary antibody detects a protein at the expected molecular weight is not enough. It’s important to show that binding is specific to the target protein, preferably by more than one method. An excellent set of recommendations has been published by the International Working Group for Antibody Validation, which lists five distinct methods, or pillars, for validating antibodies3. Four of these pillars are appropriate for validating antibodies used in Western blotting, and we’ve summarized those here.
Get low backgrounds and sensitive detection with our fluorescent secondary antibodies.
Optimized to work with Radiance Q, our HRP-conjugated secondary antibodies delivery maximum sensitivity.
Refer to azurebiosystems.com/
Note that antibody validation is application-specific—an antibody validated for ELISAs is not validated for western blotting (and vice versa) as binding occurs under distinctly different conditions.
In order to obtain an accurate measurement from a western blot, you need to ensure that the detected signal is proportional to the amount of protein present—in other words, that detection is in the linear range. However, there are multiple steps in the western blotting process that can lead to signal saturation, necessitating validation of signal linearty at each of these steps. Note that many of these steps can also be optimized to maximize the dynamic range of the signal.
To obtain the most robust quantitative western blot data, we recommend generating a standard curve that covers the full range of sample amounts you will assess, testing multiple replicates for each amount (Figure 1). A plot of signal intensity versus sample amount will indicate how much protein you can load and still be confident that your signal is in the linear range.
Note that the linear range of your system should be determined for each antibody-protein pair.
▸ I M P O R TA N T !
For the most reliable western blot quantitation, we recommend imaging all samples to be compared on the same blot, using the same exposure time, and without stripping and reprobing. While normalization can be used to bridge the different conditions, there’s always the risk of introducing variation that impacts both accuracy and precision, such as non-uniform stripping of the blot.
The amount of protein a membrane can hold can saturate. We recommend testing several transfer times and choosing the shortest time that leads to a linear signal. If the signal remains saturated at all transfer times tested, it is likely that a different step is contributing to signal saturation. In this situation you can continue optimizing other parameters and then return to transfer time and verify that membrane capacity is not saturated.
Find incubation trays and more at azurebiosystems.com/.
▸ D I D YO U K N O W ?
Today’s digital imagers and scanners can detect chemiluminescent signal over a much wider dynamic range than film, and the most powerful scanners can provide signal detection at the same sensitivity as film.
We recommend testing different antibody dilutions against the full range of target protein amounts you expect to detect. This will ensure that your assay is sensitive enough to detect the lowest amount of target protein, and has a wide enough dynamic range that detection of your highest amount of protein is still in the linear range (Figure 2).
Get sensitive, long-lasting chemiluminescent detection with Radiance ECL HRP substrate.
▸ Azure Catalog Number AC2204
The last step to assess for linearity is the exposure time of the blot to the detection system, whether that’s film or a digital imager or scanner. While exposure times need to be long enough to detect the lowest amount of protein, the exposure should be short enough that the highest amount of protein detected is still in the linear range.
Chemiluminescence can be quantitative with the proper controls and the right ECL substrate. Choose a substrate with a wide dynamic range, sensitive detection, and a long half-life for the most reliable and reproducible quantitation.
Film is typically linear over a range of up-to 1 log, a fairly narrow dynamic range. In contrast, Photomultiplier tubes can deliver linear detection over approximately 6.3 logs.
A recent study has uncovered marked molecular heterogeneity in different HeLa cell isolates at the genome, transcriptome, and proteome levels, highlighting the hazards of normalizing to a specific protein versus total protein7.
Normalization is critical for reliable western blot quantitation—by finding a feature that is expected to remain invariant across samples and conditions you can control for variations in sample preparation, loading, and transfer, and generate a relative measurement of your target protein to that invariant feature.
One widely-used method has been to normalize the signal of your target protein to that of a housekeeping protein such as GAPDH, tubulin, or actin, which was assumed to have stable and invariant levels of expression. A series of recent studies have shown how flawed this assumption can be, and now many western blotting experts and scientific journals, such as The Journal of Biological Chemistry, are recommending that western blots are either normalized to total protein instead of a housekeeping protein, or that the protein used for normalization is validated as having stable expression using the same cells and conditions as the test samples2,4,5,6.
There are different total protein stains (TPS) to choose from and the TPS you choose will affect the complexity of the total protein normalization (TPN) workflow (for a nice overview of TPS stains, see Mortiz5). Important considerations when choosing a TPS include:
For the simplest TPN workflows, we recommend using a fluorescent stain that will enable simultaneous detection of the TPS and target protein (or proteins), although this will require a fluorescent detector with two or more channels. Some TPSs are also compatible with chemiluminescent detection.
Carefully and stringently following an established protocol helps reduce variability and improve measurement precision for robust quantitative western blots. Not only should you reduce variability by using the same reagents or implementing lot-to-lot controls, keeping your incubation and wash times the same will also help ensure consistent and reliable results.
Normalizing to total protein can be just as straightforward as normalizing to a housekeeping protein. In this example, we demonstrate TPN using AzureRed Total Protein Stain to normalize signal from three different proteins found in HeLa cell lysate—tubulin, ẞ-actin, and GAPDH.
Easily stain total protein for the most accurate western blot normalization.
▸ Azure Catalog Number AC2124
See bands that other substrates miss and quantify even low-intensity signal thanks to Radiance Plus’s high signal-to-noise ratio and wide dynamic range. The most sensitive HRP substrate available from Azure, Radiance Plus delivers attomole sensitivity and a long-lasting signal.
▸ Azure Catalog Number AC2103
Get everything you need for generating consistent western blots in one convenient and easy-to-use kit. We handle the quality control of the reagents so you can stay focused on your science.
▸ Azure Catalog Number AC2140 ▸ Azure Catalog Number AC2141 ▸ Azure Catalog Number AC2104
Getting quantitative data from your western blot takes more than just a fluorescent or chemiluminescent secondary and a digital imager. You need to consider your sample prep, your normalization method, and the linearity of your signal. To help keep your quantitative western blot workflows on track, we’ve created this checklist to ensure your western blot quantitation is as accurate as it can be.
□ Have you treated your samples consistently, ensuring cells and tissues are thoroughly lysed, immediately placed on ice, and/or been treated with appropriate reagents such as protease inhibitors?
□ Have you loaded similar amounts of total protein in each lane (within a factor of 2- to 5-fold)?
□ Are you using a robust and consistent western blotting protocol (reduce variability by using the same reagents for each blot or implementing lot-to-lot controls, and keeping your transfer conditions and incubation and wash times the same)?
□ Are you normalizing to total protein?
□ Have you validated the specificity of your antibodies?
□ Have you verified that your system is linear? The linearity of your signal can be affected by multiple factors. Here are steps you can take to ensure linearity and improve the accuracy of your quantitation:
□ Are the highest and lowest amounts of your protein-of-interest in the linear range (note that you need to verify linearity with each protein-antibody pair)?
□ Are you saturating your membrane?
□ Have you optimized your primary and secondary antibody dilutions? Titrating your antibody against your protein-of-interest can reveal an antibody dilution that delivers the widest dynamic range.
□ Have you optimized your image acquisition time to ensure you are in the linear range of your imager?
Azure Imagers
Blending powerful performance with a budget-friendly price, Azure Imagers excel at quantitative western blot imaging and a whole lot more.
See the many Azure Imager configurations and get a quote at azurebiosystems.com/
Sapphire™ Biomolecular Imagers
For the ultimate in quantitative western blot imaging performance, turn to the Sapphire Biomolecular Imager.
Find out just how much performance you can pack in a hybrid laser scanner/ imager at azurebiosystems.com/
AzureRed Total Protein Stain
Easily stain total protein for the most accurate western blot normalization.
▸ Azure Catalog Number AC2124
While some view Western blotting as an art, you don’t need to be a scientific Seurat to generate consistently good Westerns. Attention to the little details can help. In this troubleshooting guide, we review some common Western blotting problems, their causes, and ways to overcome them so that you can turn your Western blot woes into Western blot wows.
CAUSE | SOLUTION |
|---|---|
Uneven protein transfer from the gel to the blotting membrane. | When assembling the blot, ensure that the transfer stack fits tightly together. You may need to change the sponge pads to improve the tightness of the fit. |
CAUSE | SOLUTION |
|---|---|
Incomplete blocking. | Change your blocking buffer. Many commonly used blocking buffers can mask the epitopes on your target, making them difficult to detect. Others may outright fail to block non-specific binding to irrelevant lysate proteins. Using an engineered blocking buffer instead of milk can reduce non-specific binding, while enhancing antibody-antigen interactions. |
Primary antibody has low-specificity for your protein-of-interest. | Run additional purification steps on your primary antibody or generate new antibody. |
Using too high of a concentration of primary antibody can reduce its specificity, leading to the appearance of off-target bands. | Increase the dilution of your primary antibody and increase the incubation time, if necessary, to offset any reduction in signal. Performing the primary antibody incubation step at 4°C can help decrease non-specific binding of your antibody. |
CAUSE | SOLUTION |
|---|---|
You might have cellular debris and/or DNA in your sample. | Change how you prepare your sample by:
|
Your loading buffer might not be well buffered or have too much salt. | Try preparing fresh loading buffer, use a different type of buffer, or reduce the salt concentration of your sample buffer through dilution, dialysis/micro-dialysis, or chromatographically. |
You may be running your gel too fast, causing the gel to heat up, degrading the polymer. | Set an appropriate maximum current and keep the voltage low. |
Your gel running buffers might not provide enough buffering capacity. | Select the right buffer for your sample. |
Buffer System | pH | Advantages |
|---|---|---|
Tris-glycine | Up to 9.5 | Good for mid-ranged proteins; inexpensive |
Tris-Bis | 6.4 | Sharp protein bands; two running buffer options for optimization based on MW |
Tris-acetate | 7.0 | High resolution of large MW proteins |
Tris-tricine | Up to 9.5 | Tricine separates low MW proteins from free SDS |
Sometimes you need to detect multiple proteins but a multiplex fluorescent western blot is just not in the cards (we know, we’ve been there!). That’s when you need a robust stripping protocol and reliable reagents. Which is why we developed our HRP Stripping buffer.
Designed to enable uniform removal of antibodies from already-developed HRP western blots, our HRP Stripping Buffer helps you strip and reprobe your western blot with confidence.
▸ Azure Catalog Number AC2154
CAUSE | SOLUTION |
|---|---|
Incomplete stripping before reprobing. | Optimize your stripping conditions by either increasing the length of time your blot is incubated in the stripping buffer or changing buffers. You also need to ensure that the blot is completely submerged in the stripping buffer and has sufficient agitation to move the buffer completely over the top of the blot. In addition, some protein-antibody pairs are more resistant to stripping than others, so the conditions that work for one study might not work for another. Another solution to consider is multiplexing detection by using fluorescently-labeled secondary antibodies. With fluorescent antibodies, you can detect as many as four different signals depending on your system. |
CAUSE | SOLUTION |
|---|---|
Air trapped between the blotting membrane and the gel. | When assembling your blot, take care to completely remove any gaps or air trapped between the blotting membrane and gel. You can use your gloved hand, a roller, or a sufficiently long glass or plastic pipet to gently roll out any air caught between the membrane and the gel. |
Insufficient contact between the primary antibody and the blot. If there is not enough primary antibody incubation solution to completely cover the blot and/or if the primary antibody solution does not move across the surface of the blot, regions of the blot may remain unexposed to the antibody. | Increase the volume of primary antibody incubation solution and verify that the solution covers all parts of the membrane during the incubation step. While you should use a small incubation tray to reduce antibody waste, the tray should large enough that the membrane can move around. |
Chemiluminescent substrate for quantitative western blots
Not all ECL substrates are created equal, and some are better for quantitative chemiluminescence than others. The ideal substrate should have a long half-life, a wide dynamic range, and offer sensitive detection. Radiance Q possesses all these features for quantitative chemiluminescent western blots that you can literally count on.
Order Radiance Q and see the performance for yourself.
▸ Azure Catalog Number AC2101
Enhance signal and lower background with this ready-to-use, 1x blocking buffer.
▸ Azure Catalog Number AC2148
CAUSE | SOLUTION |
|---|---|
Not enough sample. | Increase the amount of sample or antibody used. However, increasing reagents is not always possible, such as when you have a limited availability. There are additional ways to further optimize detection:
|
CAUSE | SOLUTION |
|---|---|
Insufficient washing. | Increase the number of washes or the length of the washing time. Additionally, increasing the amount of detergent or using a stronger detergent, such as SDS or NP-40, can further reduce background noise. |
Incomplete blocking. | Change your blocking buffer. Many commonly used blocking buffers can mask the epitopes on your target, making them difficult to detect. Others may outright fail to block non-specific binding to irrelevant lysate proteins. Using an engineered blocking buffer instead of milk can reduce non-specific binding, while enhancing antibody-antigen interactions. |
continued…
CAUSE | SOLUTION |
|---|---|
Using too high of a concentration of primary antibody can reduce its specificity, leading to general binding to the membrane. | Increase the dilution of your primary antibody and increase the incubation time, if necessary, to offset any reduction in signal. Performing the primary antibody incubation step at 4°C can help decrease non-specific binding of your antibody. |
Formulated to lower background noise and stabilize fluorescent signal.
▸ Azure Catalog Number AC2190
CAUSE | SOLUTION |
|---|---|
Nitrocellulose membranes and some types of PVDF membranes can autofluoresce, leading to a high background signal. | Switch to a low-fluorescence PVDF membrane. |
Imaging a wet membrane. | Dry the membrane with methanol before imaging. When the blot dries there is less autofluorescence from the membrane and the specific signal from the fluorophore becomes brighter. Drying the blot with methanol amplifies this effect by removing more water than air-drying alone. |
Using too high of a concentration of primary antibody can reduce its specificity, leading to general binding to the membrane. | Increase the dilution of your primary antibody and increase the incubation time, if necessary, to offset any reduction in signal. Performing the primary antibody incubation step at 4°C can help decrease non-specific binding of your antibody. |
Insufficient washing. | Increase the number of washes or the length of the washing time. Additionally, increasing the amount of detergent or using a stronger detergent, such as SDS or NP-40, can further reduce background noise. |
CAUSE | SOLUTION |
|---|---|
Loaded too much marker. | Increase the dilution of your marker. Most molecular weight ladders autofluoresce in the IR 700 channel, which is convenient for determining the molecular weight of bands in a fluorescent western blot. However, if you’re used to running chemiluminescent Western blots, you may be overloading your marker lane—we recommend running approximately one-tenth the amount of molecular weight marker on a fluorescent Western blot compared to the amount you would run on a chemiluminescent Western blot. If you can see strong bands on your membrane after transfer, you have probably loaded too much. You can also try covering the lane with the strong bands. Sometimes, it’s too late, and you can’t go back in time and load less ladder. In this case, we recommend covering up the bands with a non-fluorescent, clean, opaque material. |
