Lysate to Quantitative Western Blot Workflow
Fragmented protocols contribute to inconsistent blots, low reproducibility, and merely qualitative data. With SERVA and LICORbio’s validated Western blot bundles, you will eliminate vendor fragmentation, reduce wasteful repeat experiments, and accelerate time-to-result. Our integrated Western blot workflow solutions will give you complete confidence in your data from extraction, separation, and transfer through detection, quantification, and data analysis.
1. Extraction and Sample Preparation
A successful quantitative Western blot always begins with proper cell or tissue lysis and preparation of your sample proteins. This step involves choosing a lysis buffer based on your protein of interest and adding phosphatase or protease inhibitors as needed. We offer several lysis buffer and extraction kit options to help you get started.
SERVA RIPA Buffer
SERVA’s RIPA Buffer is an effective buffer for lysis of cultured mammalians cells. It enables protein extraction from cytoplasmic, membrane, and nuclear proteins. This buffer is compatible with many applications including protein purification, Western blotting, and reporter assays. However, it will disrupt protein-protein interactions and may be less suitable for applications like immunoprecipitation and pull-down assays.
SERVA Protein Extraction Kits
Protein extraction kits provide a convenient means of extracting various proteins. For instance, SERVA’s Mammalian Protein Extraction Kits offer a fast and efficient method for extracting total proteins from mammalian cells and tissues without ultracentrifugation. These kits contain ready-to-use, optimized extraction buffers and a protease inhibitor cocktail in a gentle formulation that preserves protein activity for downstream assays.
SERVA’s lysis buffers allow for mild but efficient lysis and high protein yield, with performance optimized and validated for several cell lines and tissues without the need for mechanical cell disruption. This single formulation also provides enhanced versatility, as protease or phosphatase inhibitors, reducing agents, and other reagents may be added prior to cell lysis. SERVA’s protein extraction kit reagents are compatible with standard protein quantification assays like immunoprecipitation, SDS-PAGE, Western blot, ELISA, EMSA, BCA protein assay, and other functional assays.
2. Electrophoresis and Protein Separation
After sample preparation and before transfer, sample proteins must be separated. This is generally performed via polyacrylamide gel electrophoresis (PAGE) or similar methods such as native PAGE or SDS-PAGE. With decades of expertise in electrophoresis, SERVA offers several user-friendly products and bundles to support this stage of the workflow.
BlueVertical™ PRiME™ Electrophoresis Chamber
The SERVA BlueVertical™ PRiME™ is a dual mini-tank system that can accommodate one or two ready-to-use gels with external dimensions of 10 x 10 x 0.7 cm. The gel assembly closes tightly and is held together by a leak-free inner core unit with a unique clamp system, and the easy-to-use BlueVertical PRiME Blot Module turns your chamber into a tank blotter.
SERVAGel™ TG PRiME™ Mini Gels
SERVA offers ready-to-use SERVAGel TG PRiME precast mini gels for quick and efficient sample separation. SERVAGel precast vertical gels are ready-to-use, individually sealed, mini polyacrylamide gels cast into unbreakable plastic cassettes. They offer superior separation performance, excellent staining and destaining properties, and overall ease of use.
- Premium resolution and superb band sharpness
- Easy and safe to operate with no leakage
- Ready-to-use with short setup times
- Unbreakable and recyclable plastic cassette
- Minimal health risk due to polymerized acrylamide and no toxic monomer
SERVAGel™ Neutral HSE Mini Gels
SERVAGel Neutral HSE high-speed electrophoresis gels provide several valuable benefits. Due to their lower acrylamide concentration, these gels consistently deliver run times under 20 minutes. They also feature an extended shelf life of 15 months, and they require no special solutions or equipment to get started.
Sample Loading Buffer
LICORbio provides 4x Protein Sample Loading Buffer that is optimized for use with Odyssey Imaging Systems. This orange loading buffer does not fluoresce in the 700 nm channel like blue loading buffers do. It is optimized for use with SDS-PAGE and Tris-Glycine-SDS running buffer, as well as Bis-Tris protein gels and MES SDS running buffer.
Protein Ladders or Markers
SERVA offers the SERVA Triple Color Protein Standard I, SERVA Triple Color Protein Standard II, and the SERVA Triple Color Protein Standard III, as well as the Quadro Color Protein Standard I and Quadro Color Protein Standard II for SDS PAGE and Western blotting.
LICORbio offers various Chameleon® Protein Ladders for fluorescence and chemiluminescence. For example, the Chameleon Duo Pre-stained Protein Ladder provides multi-colored, pre-stained bands for visual inspection and two-color near-infrared detection. Simply refer to the table below to decide which LICORbio protein ladder to choose.
SDS PAGE Bundle
Are you ready to kickstart your electrophoresis workflow? Check out our SDS PAGE Bundle! This convenient kit includes the gels, buffers, and protein marker you need to get started today.
Save over 70,00 € with our SDS PAGE Bundle that includes:
3. Membrane Transfer (Blotting)
After electrophoresis, separated proteins are generally transferred, or blotted, from the gel to a PVDF or nitrocellulose membrane via a tank, semi-dry system, or rapid-transfer system. SERVA provides various power supplies, blotters, membranes, and validated transfer reagents to support this step of the Western blot workflow.
BluePower™ Power Supply for Blot Applications
The SERVA BluePower™ 300 BLOT power supply (300 V, 2 A, 300 W) is suitable for applications requiring high current, such as tank or semi-dry blotting of larger protein gels. Four individual devices can be connected in parallel, and they are easily programmable with up to 9 steps.
BlueBlot Semi-Dry Blotter
The SERVA BlueBlot semi-dry blotter forms a homogeneous electrical field that guarantees fast and efficient transfer of proteins from gel to membrane. The anode is made from a platinum-covered steel net, and the cathode is constructed with stainless steel. Not only is the stable acrylic housing easy to clean, but also the durable electrodes can be dismounted from the housing with a flick of the wrist and cleaned under running tap water. The BlueBlot Semi-Dry Blotter is available in three models. BlueBlot SD11 accommodates one gel, BlueBlot SD17 can hold four gels, and BlueBlot SD26 holds six mini gels.
Transfer Buffers and Xpress Blotting Kits
In addition to the Towbin buffer for small proteins and the Semi-Dry Blotting Buffer Kit (discontinuous buffer system), SERVA has developed various Xpress Blotting Kits. Each kit contains ready-to-use buffers and blotting fleece (80 mm x 85 mm). The buffer reduces the transfer time, and the blotting fleeces diminish interferences during transfer.
SERVA Xpress Blotting Kits allow fast and efficient semi-dry transfer of proteins in only 15 minutes. With the ready-to-use SERVA Xpress Blotting Buffer, the efficient simultaneous semi-dry blotting of high and low molecular weight proteins is possible. The use of SERVA’s newly developed Blotting Fleece instead of blotting paper enables an undisturbed transfer in a short time. The buffer system is compatible with nitrocellulose and PVDF membranes. Any semi-dry blotter with a capacity of 400 mA can be used. The kit includes 250 mL 10X SERVA Xpress Blotting Buffer and 20x Blotting Fleece sheets. Kits including additional precut NC or PVDF membranes (10x, 80 mm x 85 mm) are available.
Choose between the Xpress Blotting Kit to use your own membranes or a kit with pre-cut membranes (Xpress NC Blotting Kit or Xpress PVDF Blotting Kit).
Nitrocellulose Membranes
SERVA nitrocellulose membranes are suitable for Western, Southern, and Northern blots. These membranes are ideal because they provide both high binding capacity and low background. The fleece-backed nitrocellulose membranes are a good alternative, since they’re easy to cut, allowing for multiple hybridizations and automatic immobilization.
Polyvinylidene Fluoride (PVDF) Membranes
PVDF membranes are hydrophobic, highly flexible, and tear resistant, and they have excellent mechanical stability. Plus, they feature high binding capacity and low background and are compatible with most staining methods. We recommend Fluorobind membranes to detect proteins with immunological or fluorescence applications.
Blotting Bundles
SERVA offers several blotting bundles to support your electrophoresis workflow. Skip the guesswork and save big on essential blotting reagents and consumables with these convenient kits. For chemiluminescence, the Tank Blot Bundle has everything you need to get started. For near-infrared or visible fluorescence, consider either our Semi-Dry or Xpress Western Bundle with your choice of IRDye® or VRDye™ Secondary Antibody.
Save 140,00 € with our Tank Blot Bundle that includes:
- Towbin Buffer for Western Blotting
- NC 0.45 Nitrocellulose Membrane, 0.45 µm, 80 × 85 mm, 10 sheets
- 2× Connection paper, 80 × 85 mm, 10 sheets
- BlueBlock PF (10x)
- WesternSure® Pro ECL Substrate 250 mL
- WesternSure® Pre-stained Chemiluminescent Protein Ladder 250 µL
Save 121,00 € with our Semi-Dry Bundle that includes:
- Semi-Dry Blotting Buffer Kit
- NC 0.45 Nitrocellulose Membrane, 0.45 µm, 80 × 85 mm, 10 sheets
- 6× Connection paper, 80 × 85 mm, 10 sheets
- Intercept® (TBS) Protein-Free Blocking Buffer 500 mL
- LICORbio Secondary Antibody of Choice
Save 139,00 € with our Xpress Western Bundle that includes:
4. Chemistry and Post-Processing of Western Blot Membranes
The accuracy, integrity, and reproducibility of your blot depend on your choice of buffers and detection chemistry. For accurate quantification, signals need to be proportional to the amount of protein loaded. While chemiluminescence can produce qualitative data, fluorescence detection is the best way to get proportional signals and quantitative protein data. SERVA and LICORbio offer many buffers, kits, substrates, and antibodies to support this critical stage of your Western blot workflow.
BlueBlock PF
Protein-free, polymer-based blocking reagent for fast blocking without loss of sensitivity in colorimetric, chemiluminescence, and fluorescence detection systems. With BlueBlock, the specific binding sites remain accessible while nonspecific reactions are suppressed, thus leading to an increase in signal intensity.
Intercept® Blocking Buffers
Intercept Blocking Buffers are formulated to provide highly efficient blocking and low background variability for replicable quantitative Western blots and other immunoassays. Available in TBS, PBS, and Protein-Free formulations from LICORbio.
Intercept Antibody Diluents
Intercept T20 Antibody Diluent improves the specificity of the primary and secondary antibodies, reducing off-target effects. The diluent contains Intercept Blocking Buffer in either tris-buffered saline (TBS) or phosphate-buffered saline preformulated with Tween® 20. Since there’s no need to mix the diluent yourself, it saves you time and reduces potential variation.
Revert® Total Protein Stains for Normalization
Make your Western blot normalization more accurate and reliable with Revert Total Protein Stain, a membrane-based (post-transfer) normalization strategy that stains all protein in your sample. Available in 700 and 520 nm formulations, Revert Total Protein Stain works with your existing Western blot workflow and does not require special gels or equipment.
SERVALight CL HRP Substrate Kits
SERVALight ready-to-use chemiluminescence substrates detect immobilised proteins (Western blot) or nucleic acids (Southern and Northern blot) either directly with HRP or indirectly with HRP-labelled antibodies/streptavidin. The SERVALight substrates are quick and easy to prepare—simply mix solution A (luminol/enhancer solution) and solution B (stabilised peroxide solution) in a 1:1 ratio.
- SERVALight Vega and SERVALight Polaris produce normal-intensity light intensity for a long duration.
- SERVALight Eos and SERVALight EosUltra produce high-intensity light for a very long duration.
- SERVALight Helios produces extremely intense light for only a brief duration.
SERVALight PreMix Substrate Kits
SERVALight PreMix Chemiluminesce HRP substrates are ready-to-use solutions for convenient and rapid detection of proteins in Western blotting. These pre-mixed solutions save time and increase the consistency of your results by avoiding pipetting errors and possible contamination.
- SERVALight PreMix Vega (mid picogram)
- SERVALight PreMix Eos (mid femtogram)
- SERVALight PreMix Helios (low femtogram)
Western Blot Staining Reagents
SERVAColor BCIP/NBT and SERVAColor TMB blot solutions are ready-to-use and non-toxic. They are used as highly sensitive, colorimetric detections of alkaline phosphatase (AP) or HRP in membrane assays. They are characterised by rapid precipitation, low background staining, long stability at room temperature, and low fading.
WesternSure® ECL Substrates
WesternSure Pro ECL Substrate offers enhanced sensitivity and prolonged signal output, making it an excellent choice for both film and imaging system applications. Its broad linear dynamic range enables precise quantification of proteins across a wide range of concentrations in a single experiment, while its high sensitivity allows detection down to the mid-femtogram range.
WesternSure Eco ECL Chemiluminescent Substrate is a highly sensitive, enhanced substrate for detecting horseradish peroxidase (HRP) on immunoblots. It is designed for high sensitivity and long signal duration, making it ideal for use with imaging systems and film. This substrate provides stable, high light intensity and allows the detection of low-abundant proteins down to the mid-femtogram detection level. This single-use formulation reduces reagent contamination and water consumption.
Primary and Secondary Antibodies
IRDye Secondary Antibodies are perfect partners for Odyssey Imagers and the ideal choice for quantitative Western blots. They provide the highest sensitivity due to low background autofluorescence in the near-infrared and the highest signal-to-noise ratios. IRDye Secondary Antibodies are also highly cross-adsorbed by default, maximizing specificity, minimizing non-specific binding, and supporting multiplex detection and quantification of multiple targets. Available in various formulations and conjugations directly from LICORbio. LICORbio also offers anti-rabbit and anti-mouse HRP-labeled secondary antibodies, as well as several primary antibodies for A-Tubulin, B-Tubulin, B-Actin, Cox IV, GAPDH, and Vinculin.
Stripping Buffers
BlueClear SB is a ready-to-use buffer for efficient stripping of high-affinity antibodies from Western blot membranes after chemiluminescence detection. After incubating the membrane in the stripping buffer for 30–60 min at room temperature, difficult-to-remove antibodies are efficiently stripped by incubation in the heated buffer. After washing in PBST or TBST, the membrane can be blocked again and incubated with antibodies.
LICORbio offers WesternSure Stripping Buffer and NewBlot™ Stripping Buffers for ECL, PVDF, nitrocellulose, and IR applications.
5. Detection and Quantification
For a Western blot to yield quantitative data, the signal must be proportional to the amount of sample loaded. The relationship between the amount of protein on the blot and the measured signal can only be known within the linear range of detection.
Targets shouldn’t be quantified or compared outside the linear range of detection. High sensitivity without saturation is the key to a wide linear dynamic range, and a wide dynamic range makes it easier to produce Western blot data within the linear range.
Odyssey® Imaging Systems
LICORbio® Odyssey® Imaging Systems are the gold standard for fluorescence imaging, protein quantification, and quantitative Western blots. With 6+ logs of dynamic range, high sensitivity and no saturation, and over 85,000 citations in peer-reviewed publications, Odyssey Imagers have become synonymous with reliable quantification of Western blots, 2D cell culture, and immunohistochemistry.
LICORbio Atlas™ Imaging System
The LICORbio Atlas Imager is a versatile screening platform that delivers quantitative data across a wide range of assays in fluorescence, luminescence, and brightfield. Built on 30 years of trusted LICORbio technology, this instrument delivers unique screening capabilities that help you save time, money, and lab space.
6. Software and Data Analysis
Quantitative protein data analysis requires reliable, trustworthy software. LICORbio offers two industry-leading software solutions to support your quantitative Western blot workflow. Choose the one that best suits your lab’s needs, or leverage the benefits of both for specific use cases.
Image Studio™ Software
With over 150,000 downloads worldwide, LICORbio Image Studio™ Software is a trusted standard for flexible, reproducible image acquisition and analysis. Its adaptable design provides greater control over analysis parameters in a single package, without manipulating the image. It is optimized to support Odyssey Imaging Systems and the LICORbio Atlas Imager.
Empiria Studio® Software
LICORbio Empiria Studio Software takes the guesswork out of quantification. Use it to automatically normalize your target protein data based on your chosen internal loading control. Significantly reduce user-to-user variability with patented Adaptive Lane Finding and Adaptive Background Subtraction. Determine target protein expression, build a library of validated antibodies, compare replicates, and automatically calculate advanced statistical values, including % CV, standard deviation, and signal-to-noise ratio—all in one reliable software.
Frequently Asked Questions (FAQs)
What Is a Quantitative Western Blot?
A quantitative Western blot makes relative comparisons between different treatments possible. The goal of a quantitative Western is to accurately measure changes in protein expression.
What Are Common Uses of Western Blots?
- Protein-protein interactions
- Signaling pathways
- Post-translational modifications
- Cell surface proteins
- RNAi analysis
- Published examples of quantitative Western blots
Why Do We Need Quantitative Western Blots?
Life-altering therapeutics. Increased crop yields. All of us want to make a difference with our life’s work. Quantitative Westerns can be a powerful tool to advance discovery and make the world a better place.
What Are the Requirements for Quantitative Western Blots?
- Signals are proportional to the amount of protein loaded.
- Proteins of interest (or “targets”) are quantified within the linear range of detection.
- Internal loading controls correct for unavoidable variability from sample preparation, loading, and transfer.
- Along with these requirements, experimental variability should be kept low so that replication is achievable.
- For a Western to be quantitative, you must validate several conditions. A difference in band intensity can be caused by experimental variability (like using a different antibody or blocker), or by true biological changes. It’s important to determine the sources of variability in your Western blotting procedure, and how best to control for them, before you even design your experiment.
What Is Variability?
Your conclusions will always be based on (and tempered by) the amount of variability in your experiment. You might define or measure variability with CV (coefficient of variation), σ (standard deviation), r² (coefficient of determination), or something else entirely. Reducing variability is the key to maximizing precision.
Determining the significance of experimental results is a common concern for quantitative Westerns. One way is to define the percent change of band signal between treated and untreated samples. The experimental change needs to be greater than your measure of variability, like CV. So, to be more confident of small changes, decrease the CV in your experiment. To get accurate answers consistently, remove sources of error wherever possible.
Why Detect in the Linear Range?
Outside the linear range of detection, the relationship between the amount of protein on the blot and the measured signal is unknown. Your relative measurement of protein amount is then not representative of a significant, quantitative value.
Linear range is the span of signal intensities that display a linear relationship between the amount of protein on the membrane and signal intensity recorded by the detector. Dynamic range is the range of band intensities the detection system can measure in a single capture. High sensitivity without saturation is the key to a wide linear dynamic range. Targets shouldn’t be quantified or compared outside the linear range of detection. A wide dynamic range makes it easier to produce Western blot data within the linear range. An ideal detection system picks up faint signals without saturating strong signals.