Revert™ Total Protein Stain Normalization Protocol

Introduction

In quantitative Western blotting (QWB), normalization mathematically corrects for unavoidable sample-to-sample and lane-to-lane variation by comparing the target protein to an internal loading control. The internal loading control is used as an indicator of sample protein loading, to correct for loading variation and confirm that observed changes represent actual differences between samples.

For more normalization related resources, see " Further Reading".

Total Protein Staining as an Internal Loading Control

Total protein detection is becoming the “gold standard” for normalization of protein loading. After transfer, but prior to immunodetection, the membrane is treated with a total protein stain to assess actual sample loading across the blot. Because this internal loading control uses the combined signal from many different sample proteins in each lane, error and variability are minimized. This antibody-independent method corrects for variation in both sample protein loading and transfer efficiency, and monitors protein transfer across the blot at all molecular weights.

Revert™ 700 Total Protein Stain and Revert 520 Total Protein Stain (licor.com/revert) are near-infrared fluorescent membrane stains used for total protein detection and normalization. Revert staining is imaged at 700 nm or 520 nm, and fluorescent signals are proportional to sample loading.

This protocol describes how to use Revert 700 Total Protein Stain and Revert 520 Total Protein Stain for Western blot normalization and quantitative analysis.

The Odyssey M Imager is the only Odyssey Imager that supports the 520 channel.

Keys for Success

Keys for Success

  • Saturation and linear range. Saturated bands and sample overloading frequently compromise the accuracy of QWB. Use a dilution series to verify that you are working within the linear range of detection, and signal intensity is proportional to sample loading. See the protocol: Determining the Linear Range for Quantitative Western Blot Detection (licor.com/LinearRange) for more information.

  • Replication. Replicate samples provide information about the inherent variability of your methods, to determine if the changes you see are meaningful and significant. A minimum of three technical replicates is recommended for each sample. Replicates are discussed further on page Normalization Calculations and Analysis of Replicates.

  • Uniform sample loading. Uniform loading of total sample protein across the gel is critical for accurate QWB analysis. A protein concentration assay (BCA, Bradford, or similar assay) must be used to adjust sample concentration and load all samples as consistently as possible.

    You can use reagents designed to confirm uniform sample loading, such as Odyssey Loading Indicators (P/N 926-20002), to improve the accuracy of this validation protocol. However, these reagents do not preclude the need to perform a protein concentration assay before sample preparation and loading.

  • Antibody validation. Two-color Western blot detection requires careful selection of primary and secondary antibodies to prevent cross-reactivity. Always perform single-color control blots first to verify antibody specificity, and to identify possible interference from background bands.

    The Antibody Publication Database can help you find antibody pairs that work for your experiment (licor.com/antibodyrequest).

  • Antibody validation. Verify specificity of the phospho-antibody to ensure that it does not cross-react with the unmodified target protein, and to identify possible interference from background bands. Important guidelines are provided in Section

    The Antibody Publication Database can help you find antibody pairs that work for your experiment (licor.com/antibodyrequest).

  • Phosphorylation stoichiometry. This protocol is intended for relative comparison of pan-protein and phospho-protein signals, and results do not indicate the stoichiometry of phosphorylation (1).

Required Reagents

  1. Treated and untreated samples

    Protein concentration must be determined for all samples.

  2. Revert™ 700 Total Protein Stain Kit and Revert 520 Total Protein Stain Kit (licor.com/revertkit)

    Revert 700 Total Protein Stain and Revert 520 Total Protein Stain are used to assess sample protein loading in each lane as an internal loading control. After transfer and prior to immunodetection, the membrane is treated with this near-infrared fluorescent protein stain and imaged. Membrane staining can verify that sample protein was uniformly loaded across the gel, and assess the quality and consistency of protein transfer.

  3. Odyssey Loading Indicator, 800 nm (LI-COR, P/N 926-20002)

    Odyssey Loading Indicator (OLI) is an external loading control that is added to your samples just before electrophoresis and is used to verify that a similar sample volume was loaded in each lane. Because it is an exogenous protein, it does not provide information about the amount of sample protein loaded or transferred.

  4. Electrophoresis reagents

  5. Transfer reagents

  6. Pan-specific and modification-specific antibodies against target protein

    Antibody selection and validation are described in Section

  7. Western blot detection reagents (near-infrared fluorescence)

    Perform near-infrared Western blot detection according to the Near-Infrared Western Blot Detection Protocol (licor.com/NIRWesternProtocol; LI-COR).

Protocols

These protocols highlight the main methods and steps for using Revert™ 700 Total Protein Stain or Revert 520 Total Protein Stainfor Western blot normalization and quantitative analysis.

Prepare and Transfer Proteins

  1. Generate a set of experimental samples (drug treatment, time course, dose-response, etc).

    A minimum of three replicates should be performed for each sample.

  2. Determine the protein concentration of each sample using a BCA, Bradford, or similar protein assay.

  3. Dilute the samples to equal concentrations to enable consistent, uniform loading of total sample protein across the gel.

  4. Prepare samples to be loaded on the gel with sample loading buffer.

  5. Denature sample by heating at 95 °C for 3 minutes (or 70 °C for 10 minutes).

  6. Load a uniform amount of sample protein in each lane.

  7. Separate sample proteins by SDS-PAGE.

  8. Transfer proteins to blotting membrane.

Using Revert 520 Total Protein Stain

Use the steps below to stain using Revert 520 Total Protein Stain and to detect targets in the 700 nm and 800 nm channels.

You will image the membrane on an Odyssey M Imager or Odyssey F Imager twice in this protocol: once in step 2 and once in step 5. The first acquisition will be of Revert 520 Total Protein Stain by itself. You will then remove the total protein stain and acquire an image of your targets in the 700 and 800 nm channels.

Method 1: Total Protein Detection Before Western Blot Detection

Step 1. Stain with Revert™ 520 Total Protein Stain
  1. Add methanol to the stain reagents as indicated on each bottle.

  2. After transfer is complete, fully dry the membrane. Place the membrane on top of a piece of clean filter paper and allow it to dry:

    • 40 to 60 minutes at room temperature.

    • 10 minutes in an oven at 37 °C.

    • Overnight at room temperature as a stopping point.

  3. Rehydrate the membrane after fully drying.

    • For nitrocellulose membranes, incubate the membrane in TBS or PBS (no detergent) for 5 minutes at room temperature with gentle shaking.

    • For PVDF membranes, first rehydrate using 100% methanol for 30 seconds. Then rinse in TBS or PBS (no detergent) for 5 minutes at room temperature with gentle shaking.

    4. Do not allow the membrane to dry from this point on.

  4. Rinse the membrane with ultrapure water.

    5. Before moving to the next step, ensure the membrane container provides a minimum clearance of 1/8th of an inch on all sides. Revert 520 Total Protein Stain will cause the membrane to swell. Without clearance, staining may be uneven.

  5. Stain the membrane with Revert 520 Total Protein Stain. Incubate the membrane in 10 mL of Revert 520 Total Protein Stain solution for 5 minutes at room temperature with gentle shaking.

    6. For more information about the Revert protocol, see licor.com/revert.

  6. Decant the total protein stain solution thoroughly. Using approximately 10 mL of Revert Wash Solution for each wash, rinse the membrane two times for 30 seconds at room temperature with gentle shaking.

  7. Decant the wash solution thoroughly, then briefly rinse the membrane with ultrapure water.

Step 2. Acquire Revert 520 Total Protein Stain Image
Step 3. Destaining

The destaining procedure must be performed for Revert 520 Total Protein Stain.

  1. After the first imaging, briefly rinse the membrane with ultrapure water.

    This step is important for the destaining step to work properly.

  2. Incubate the membrane in 10 mL of Revert Destaining Solution for 5 to 10 minutes, with gentle shaking. Destaining is complete when stain is no longer visible by eye.

    Do not destain for longer than 10 minutes.

  3. Decant the destaining solution thoroughly, then briefly rinse the membrane with ultrapure water.

    Proceed immediately to blocking and immunodetection.

Step 4. Proceed with Western Blot Protocol

Follow your normal Western blot protocol using IRDye® 800CW Secondary Antibody to detect your target in the 800 nm channel and IRDye 680RD Secondary Antibody to detect your target in the 700 nm channel.

It is recommended to use 700 nm channel detection for your most abundant target and the 800 nm channel for weak or low abundance targets.

Step 5. Acquire Image for Target Channel(s)

Image the membrane in the 700 and 800 nm channels. Instructions for imaging are provided in Imaging the Blot in LI‑COR® Acquisition Software.

After destaining and Western blot processing, residual fluorescence may be detected in the 520 and 700 nm channel of less than 2%.

Method 2: Total Protein Detection After Western Blot Detection

This method is useful if total protein staining is desired, but not performed on the membrane prior to Western blot detection.

You will image the membrane two times in this protocol, once in step 2 and once in step 5. The first acquisition will be of the target proteins in the 700 and/or 800 channels. The second acquisition will be of the Revert 520 Total Protein Stain.

This method only works if the membrane is processed with a protein-free blocking buffer and antibody diluent.

Step 1. Process Western Blot with Protein-Free Antibody Diluent and Blocking Buffer

Block membrane in Intercept® Protein-Free Blocking Buffer and use Intercept Protein-Free Antibody Diluent for primary and secondary antibody incubations.

Step 2. Acquire Image for Target Channel(s)

Image membrane to capture 700 and 800 nm detection channels.

Step 3. Rinse Membrane with Ultrapure Water

Rinse the membrane with ultrapure water.

Before moving to the next step, ensure the membrane container provides a minimum clearance of 1/8th of an inch on all sides. Revert 520 Total Protein Stain will cause the membrane to swell. Without clearance, staining may be uneven.

Step 4. Stain with Revert 520 Total Protein Stain

  1. Add methanol to the stain reagents as indicated on each bottle.

  2. Stain the membrane with Revert 520 Total Protein Stain. Incubate the membrane in 10 mL of Revert 520 Total Protein Stain solution for 5 minutes at room temperature with gentle shaking.

    Do not allow the membrane to dry from this point on.

  3. Decant the total protein stain solution thoroughly. Using approximately 10 mL of Revert Wash Solution for each wash, rinse the membrane two times for 30 seconds at room temperature with gentle shaking.

  4. Decant the wash solution thoroughly, then briefly rinse the membrane with ultrapure water.

Step 5. Acquire Revert 520 Total Protein Stain Image

Do not allow the membrane to dry during imaging. To prevent drying, you may add ultrapure water on top of the membrane.

Immediately image the membrane in the 520 nm channel.

Revert 700 Total Protein Stain Protocols

Method 1: Single-Color Western Blot (800 nm target only)

Use the steps below for detection of a target in the 800 nm channel.

Step 6. Stain with Revert™ 700 Total Protein Stain
  1. Add methanol to the stain reagents as indicated on each bottle.

  2. After transfer is complete, fully dry the membrane. Place the membrane on top of a piece of clean filter paper and allow it to dry:

    • 40 to 60 minutes at room temperature.

    • 10 minutes in an oven at 37 °C.

    • Overnight at room temperature as a stopping point.

  3. Rehydrate the membrane after fully drying.

    • For nitrocellulose membranes, incubate the membrane in TBS or PBS (no detergent) for 5 minutes at room temperature with gentle shaking.

    • For PVDF membranes, first rehydrate using 100% methanol for 30 seconds. Then rinse in TBS or PBS (no detergent) for 5 minutes at room temperature with gentle shaking.

    Do not allow the membrane to dry from this point on.

  4. Rinse the membrane with ultrapure water.

    Before moving to the next step, ensure the membrane container provides a minimum clearance of 1/8th of an inch on all sides. Revert 700 Total Protein Stain will cause the membrane to swell. Without clearance, staining may be uneven.

  5. Stain membrane with Revert 700 Total Protein Stain. Incubate the membrane in 5 mL of Revert 700 Total Protein Stain solution for 5 minutes at room temperature with gentle shaking.

    For more information about the Revert protocol, see licor.com/revert.

  6. Decant total protein stain solution thoroughly. Using approximately 5 mL of Revert 700 Wash Solution (P/N 926-11012), rinse the membrane two times for 30 seconds at room temperature with gentle shaking.

  7. Decant wash solution thoroughly, then briefly rinse the membrane with ultrapure water.

You do not need to destain Revert 700 Total Protein Stain in this protocol as you will be visualizing your HKP and OLI in the 800 nm channel. If you wish to visualize several HKPs in the 700 nm and 800 nm channels, you can do so by destaining Revert according to the instructions in your pack insert.

Step 7. Image Membrane

Do not allow the membrane to dry during imaging. If you are using an Odyssey M, Odyssey DLx, or Odyssey F, it is best to place the silicone mat on top of the membrane. See the Operator's Manual for your imager for detailed instructions (licor.com/support).

  1. Immediately image the membrane in the 700 nm channel using an Odyssey Imaging System. If saturation occurs, reduce the scan intensity or acquisition time, or use AutoScan if your instrument includes this.

  2. Proceed immediately to blocking and follow your normal Western blot protocol using IRDye® 800CW Secondary Antibody to detect your target in the 800 nm channel.

  3. Image the membrane in the 800 nm channel with an Odyssey Imaging System. If saturation occurs, reduce the scan intensity or acquisition time, or use AutoScan if your instrument includes this.

    Visible color from stain will wash off during processing and residual total protein signal may be detected in the 700 nm channel.

Method 2: Two-Color Western Blot (700 and 800 nm targets)

Use the steps below for detection of targets in the 700 nm and 800 nm channels. Follow the single-color Western blot steps up until imaging the membrane with Revert™ 700 Total Protein Stain in the 700 nm channel (step 2.1). After imaging the membrane, proceed to the destaining step.

Step 1. Destaining

  1. Briefly rinse membrane with ultrapure water.

    This step is important for the destaining step to work properly.

  2. Incubate the membrane in 5 mL of Revert Destaining Solution (P/N 926-11013) for 5 to 10 minutes, with gentle shaking. Destaining is complete when stain is no longer visible by eye.

    Do not destain for longer than 10 min.

Step 2. Process Western Blot

  1. Decant destaining solution thoroughly, then briefly rinse the membrane with ultrapure water. Proceed immediately to blocking and immunodetection.

  2. Follow your normal Western blot protocol using IRDye® 800CW Secondary Antibody to detect your target in the 800 nm channel and IRDye 680RD Secondary Antibody to detect your target in the 700 nm channel.

    It is recommended to use 700 nm channel detection for your most abundant target and the 800 nm channel for weak or low abundance targets.

  3. Image the membrane in the 700 and 800 nm channels with an Odyssey Imaging System. If saturation occurs, reduce the scan intensity or acquisition time, or use AutoScan if your instrument includes this.

    After destaining and Western blot processing, 1-3% residual fluorescence from Revert may be seen during imaging in the 700 nm channel, but this will not impact results.

Method 3: Total Protein Detection After Western Blot Detection

This method is useful if total protein staining is desired, but not performed on the membrane prior to Western blot detection.

You will image the membrane two times in this protocol, once in step 2 and once in step 5. The first acquisition will be of the target proteins. The second acquisition will be of the Revert 700 Total Protein Stain.

This method only works if the membrane is processed with a protein-free blocking buffer and antibody diluent.

Step 1. Process Western Blot with Protein-Free Antibody Diluent and Blocking Buffer

Block membrane in Intercept® Protein-Free Blocking Buffer and use Intercept Protein-Free Antibody Diluent for primary and secondary antibody incubations.

Step 2. Acquire Image for Target Channel(s)

Image membrane to capture detection channels for target proteins.

Step 3. Rinse Membrane with Ultrapure Water

Rinse the membrane with ultrapure water.

Before moving to the next step, ensure the membrane container provides a minimum clearance of 1/8th of an inch on all sides. Revert 700 Total Protein Stain will cause the membrane to swell. Without clearance, staining may be uneven.

Step 4. Stain with Revert 700 Total Protein Stain

  1. Add methanol to the stain reagents as indicated on each bottle.

  2. Stain the membrane with Revert 700 Total Protein Stain. Incubate the membrane in 10 mL of Revert 700 Total Protein Stain solution for 5 minutes at room temperature with gentle shaking.

    Do not allow the membrane to dry from this point on.

  3. Decant the total protein stain solution thoroughly. Using approximately 10 mL of Revert Wash Solution for each wash, rinse the membrane two times for 30 seconds at room temperature with gentle shaking.

  4. Decant the wash solution thoroughly, then briefly rinse the membrane with ultrapure water.

Step 5. Acquire Revert 700 Total Protein Stain Image

Do not allow the membrane to dry during imaging. If you are using an Odyssey DLx or an Odyssey M, it is best to place the silicone mat on top of the membrane. See the Operator's Manual for your imager for detailed instructions (licor.com/support).

Immediately image the membrane in the 700 nm channel using the Odyssey M Imager.

Imaging the Blot in LI‑COR® Acquisition Software

Follow the instructions below to image your Western blot membrane using LI‑COR Acquisition Software.

  1. Start LI‑COR Acquisition Software LI-COR Acquisition icon.

  2. From the Getting Started page, choose Scan.

    LI-COR Acquisition scan icon

  3. On the Connect page, ensure your username is selected and choose the Odyssey M Imager or Odyssey F Imager that you want to use from the Imager list.

    Click Connect.

  4. Choose Membrane on the Choose Assay page.

    LI-COR Acquisition choose membrane

    Choosing the appropriate assay will ensure that the image is available for the correct analysis workflow in Empiria Studio® Software.

  5. Create a Scan Area for each blot that you want to image by drawing a rectangle around the location of each blot on the Scan Surface.

    LI-COR Acquisition draw scan area

    Click Next.

  6. On the Detection Options page, click the Select Channels button beside each Scan Area. For each, be sure to select the detection channels you will be imaging.

    LI-COR Acquisition select detection channel

  7. Click Scan to begin the scan.

Total Protein and HKP Quantification

Quantify the fluorescent signals from the total protein stain (700 nm or 520 nm), HKP (800 nm), and loading indicator (800 nm). An Empiria Studio® Software workflow guides you through this process step-by-step. The provided Image Studio™ Software instructions are for the 700 nm and 800 nm channels only.

To learn more about the Empiria Studio® Software workflow for this process, go to licor.com/empiria.

Target Protein and HKP Quantification

Quantify the fluorescent signals of the HKP (700 nm) and target protein (800 nm). The following instructions are for Image Studio™ Software. The Empiria Studio Software HKP Normalization workflow guides you step-by-step through the process.

To learn more about the Empiria Studio® Software workflow for this process, go to licor.com/empiria.

Total Protein and Target Quantification

Quantify the fluorescent signals from Revert staining (700 nm) and your target protein (800 nm). The following instructions are for Image Studio™ Software. An Empiria Studio® Software workflow guides you step-by-step through the process.

To learn more about the Empiria Studio® Software workflow for this process, go to licor.com/empiria.

Pan Protein and Phospho-Protein Quantification

Quantify the fluorescent signals for the pan protein (700 nm) and phosphorylated target protein (800 nm). The following instructions are for Image Studio™ Software. The Empiria Studio® Software Post-Translational Modification workflow guides you step-by-step through the process.

To learn more about the Empiria Studio® Software workflow for this process, go to licor.com/empiria.

Total Protein Quantification

The Empiria Studio® Software Linear Range Determination workflows guide you step-by-step through this process. For more detailed information, see the Linear Range Determination in Empiria Studio Software (licor.com/LinearRangePaper) white paper. The following instructions are for using Image Studio™ Software.

Before you begin, under the Analysis tab change the type to Manual. Use the Draw Rectangle tool in Image Studio Software to quantify the total protein signal in each lane.

  1. Draw a shape around the first lane on the total protein stain image (700 nm channel image).

    1. In the Shape group of the Analysis tab, click Draw Rectangle.

      Image Studio draw rectangle

    2. Draw a rectangle around the first lane.

      If lanes are skewed, rotate the shape by clicking Rotate in the Edit group.

      For help choosing the right background subtraction method, see licor.com/BgSubtractHelp.

  2. Add the shape from the first lane to each remaining lane.

    1. With the first shape selected, click Add Selection.

    2. Click the next lane to copy the shape.

      If necessary, adjust the shape borders to ensure that each shape encloses the signal from one, and only one lane.

  3. Export total protein quantification data.

    1. Click Shapes to open the Shapes data table.

      Image Studio shapes table highlighted

    2. Select shape data, then copy and paste data into a spreadsheet, or click Report to export the data into an external spreadsheet.

      All data fields will be exported, but “Signal” is the field of interest for analysis.

Revert Total Protein Stain lane boxes

Target Protein Quantification

The Empiria Studio® Software Linear Range Determination workflows guide you step-by-step through this process. For more detailed information, see the Linear Range Determination in Empiria Studio Software white paper (licor.com/LinearRangePaper). The following instructions are for using Image Studio™ Software.

Before you begin, under the Analysis tab change the type to Manual. Use the Add Rectangle tool in Image Studio Software to quantify the target bands.

  1. Click to select your Western blot target channel image, then add shapes to bands.

    1. In the Shape group of the Analysis tab, click Add Rectangle.

      Image Studio add rectangle

    2. Click each band to be analyzed, and an appropriately sized shape will be added around the band.

      Revert normalization target bands

      For help choosing the right background subtraction method, see licor.com/BgSubtractHelp.

  2. Export quantification data.

    1. Click Shapes to open the Shapes data table.

      Image Studio shapes table highlighted

    2. Select shape data, then copy and paste data into a spreadsheet, or click Report to export the data into an external spreadsheet.

      All data fields will be exported, but “Signal” is the field of interest for analysis.

Revert normalization target band boxes

Normalization Calculations and Analysis of Replicates

Replication is an important part of QWB analysis, and is used to confirm the validity of observed changes in protein levels. Biological and technical replicates are both important, but meet different needs (2, 3).

  • Biological replicates: Parallel measurements of biologically distinct samples, used to control for biological variation and examine the generalizability of an experimental observation.

  • Technical replicates: Repeated measurements used to establish the variability of a protocol or assay, and determine if an experimental effect is large enough to be reliably distinguished from the assay noise.

Technical replication can be performed by testing the sample multiple times on the same gel or membrane (intra-assay variation) or by testing the sample multiple times in several Western blot experiments. This procedure describes the normalization and analysis of technical replicates that were tested multiple times on the same membrane.

Empiria Studio® Software will perform these calculations automatically. Please continue to the Data Interpretation section.

Calculate the Lane Normalization Factor for Each Lane (Total Protein Stain, 700 nm)

  1. Prepare a spreadsheet that contains the total protein staining and target protein quantification data.

  2. Using the total protein stain data from the normalization channel, calculate the average (arithmetic mean), standard deviation, and % coefficient of variation (% CV) of the replicate samples.

    “Average” formula in Excel = AVERAGE(rep 1, rep 2, ……)

    Standard Deviation formula in Excel = STDEV(rep 1 value, rep 2 value, …..)

    Percent coefficient of variation formula

    Revert Staining (700 nm)

    LaneSample treatmentReplicate700 nm signalAverage SignalSt Dev% CV
    1none110,0009,000100011%
    2 28,000   
    3 39,000   
    4UV18,0008,3335777%
    5 29,000   
    6 38,000   

    Example values shown for illustration only.

  3. Calculate the lane normalization factor (LNF) for each lane.

    1. Identify the lane with the highest signal for total protein staining.

    2. Use this value to calculate the lane normalization factor for each lane.

      Revert Staining (700 nm)

      LaneSample treatmentReplicate700 nm signalHighest SignalLane Normalization factor
      1none110,00010,0001
      2 28,00010,0000.8
      3 39,00010,0000.9
      4UV18,00010,0000.8
      5 29,00010,0000.9
      6 38,00010,0000.8

Normalization factors must be calculated for each blot. Normalization factors and standard curves cannot be reused between blots.

Calculate the Normalized Target Protein Signals (800 nm)

  1. Using the target protein data from the 800 nm channel, calculate the average, standard deviation, and % coefficient of variation (%CV) of the replicate samples.

    Target protein (800 nm), not normalized

    LaneSample treatmentReplicateTarget (800 nm)Average SignalSt Dev% CV
    1none1650600508%
    2 2550   
    3 3600   
    4UV1450477255%
    5 2500   
    6 3480   

    Example values shown for illustration only.

  2. Calculate the normalized target signal for each target band by applying the lane normalization factor for that lane.

    1. Divide the target signal for each lane by the corresponding LNF.

      Revert normalized signal formula

      Target protein (not normalized)NormalizationNormalized to total protein
      LaneSample treatmentReplicateTarget (800 nm)LNF (Revert)Apply LNFNorm. Target
      1none16501650 / 1650
      2 25500.8550 / 0.8688
      3 36000.9600 / 0.9667
      4UV14500.8450 / 0.8563
      5 25000.9500 / 0.9556
      6 34800.8480 / 0.8600

    2. Calculate the average, standard deviation, and % coefficient of variation of the replicates.

      Target protein (not normalized)Target protein (normalized to total protein) 
      LaneSample treatmentReplicateTarget (800 nm)Normalized TargetAverage signalSt Dev% CV
      1none1650650668193%
      2 2550688   
      3 3600667   
      4UV1450563573244%
      5 2500556   
      6 3480600   

Data Interpretation

  1. Use the normalized target protein values for relative comparison of samples.

    In the example above, target protein level is 14% lower in UV-treated samples than in untreated samples.

  2. % CV can be used to evaluate the robustness of QWB results, and determine if the magnitude of observed changes in target protein levels is large enough to be reliably distinguished from assay variability.

    1. The percent coefficient of variation (% CV) describes the spread or variability of measured signals by expressing the standard deviation (SD) as a percent of the average value (arithmetic mean). Because % CV is independent of the mean and has no unit of measure, it can be used to compare the variability of data sets and indicate the precision and repeatability of an assay.

      • A low % CV value indicates low signal variability and high measurement precision.

      • A larger % CV indicates greater variation in signal and reduced precision

    2. On a Western blot, a change in band intensity is meaningful only if its magnitude substantially exceeds the % CV.

      • Generally speaking, the magnitude of the reported change should be at least 2X greater than the % CV.

        Example: To report a 20% difference between samples (0.8-fold or 1.2-fold change in band intensity), a CV of 10% or less would be recommended for replicate samples.

        For a specific measurement, this threshold for the magnitude of change would correspond to the mean + 2 SD.

      • Faint bands or subtle changes in band intensity are more difficult to detect reliably. In these situations, QWB analysis requires more extensive replication and low % CV.

      • This is a general guideline only. Replication needs and data interpretation are specific to your experiment, and you may wish to consult a statistician.

  3. Compare the % CV of Target Protein replicates before and after normalization.

    1. Normalization should not greatly increase the % CV of replicate samples.

    2. The purpose of normalization is to reduce the variability between replicate samples by correcting for lane-to-lane variation. A large increase in % CV after normalization of replicates is a warning sign that the normalization method is not sufficiently robust, and may be a source of experimental error.

References

1. Janes KA (2015) An analysis of critical factors for quantitative immunoblotting. Sci Signal. 8(371): rs2.

2. Robasky, K, Lewis NE, and Church GM. Nat. Rev. Genet. 15: 56–62 (2014). http://www.nature.com/nmeth/journal/v11/n9/pdf/nmeth.3091.pdf

3. Naegle K, Gough NR, and Yaffe MB. Sci Signal. 8:fs7 (2015). https://www.ncbi.nlm.nih.gov/pubmed/25852186

Further Reading

Please see the following for more information about QWB analysis.

  • Western Blot Normalization Handbook

    licor.com/handbook

    The Normalization Handbook describes how to choose and validate an appropriate internal loading control for normalization.

  • Good Normalization Gone Bad

    licor.com/GNGB

    Good Normalization Gone Bad presents examples of normalization that have been adversely affected by common pitfalls and offers potential solutions.

  • Western Blot Normalization White Paper

    licor.com/normalizationreview

    This white paper comprehensively reviews the literature of Western blot normalization.

  • Determining the Linear Range for Quantitative Western Blot Detection

    licor.com/LinearRange

    This protocol explains how to choose an appropriate amount of sample to load for QWB analysis.

  • Revert™ Total Protein Stain Normalization Protocol

    licor.com/RevertNormalization

    This protocol describes how to use Revert Total Protein Stain for Western blot normalization and analysis.

  • Pan/Phospho Analysis For Western Blot Normalization

    licor.com/PanProteinNormalization

    This protocol describes how to use pan-specific antibodies as an internal loading control for normalization.

  • Housekeeping Protein Validation Protocol

    licor.com/HKP-Validation

    This protocol explains how to validate an HKP for use as an internal loading control, by demonstrating that HKP expression is stable in the relevant experimental samples.

  • Housekeeping Protein Normalization Protocol

    licor.com/HKP-Normalization

    This protocol describes how to use a housekeeping protein for Western blot normalization and quantitative analysis.

  • Linear Range Determination in Empiria Studio® Software White Paper

    licor.com/LinearRangePaper

    This white paper describes how Empiria Studio Software guides the user through the linear range validation process and documents the results for future use.