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Analytical Biochemistry
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A B S T R A C T
Attaining true quantitative data from WB requires that all the players involved in the procedure are quality controlled including the user. Appropriate protein
extraction method, electrophoresis, and transfer of proteins, immunodetection of blotted protein by antibodies, and the ultimate step of imaging and analyzing the
data is nothing short of a symphony. Like with any other technology in life-sciences research, Western blotting can produce erroneous and irreproducible data. We
provide a systematic approach to generate quantitative data from Western blot experiments that incorporates critical validation steps to identify and minimize
sources of error and variability throughout the Western blot process.
∗
Corresponding author.
E-mail addresses: lakshmi.pillai@licor.com, lspi222@g.uky.edu (L. Pillai-Kastoori).
https://doi.org/10.1016/j.ab.2020.113608
Received 1 August 2019; Received in revised form 16 December 2019; Accepted 27 January 2020
Available online 31 January 2020
0003-2697/ © 2020 LI-COR BIOSCIENCES, LINCOLN, NEBRASKA 68504. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
Western blot data; the accuracy and validity of analysis are based on Adoption of HKPs as ILCs for immunoblotting was inspired by
this foundation. Because it is influenced by numerous experimental housekeeping genes, the stably expressed transcripts used as en-
factors, the linear range should be determined empirically for each dogenous controls for analysis of gene expression analysis. Stable ex-
Western Blot assay. A combined linear range denotes the linear range pression of actin, tubulin, and other housekeeping genes in many cell
for both the target protein as well as a loading protein [also called types and tissues initially indicated that their protein products should
Internal Loading Control (ILC)]. Internal loading control as the name be similarly appropriate for Western blot normalization. However, the
suggests is present internal to the test sample itself and could be a single expression of some HKPs is now known to be altered in response to
Housekeeping protein (HKP) or total cellular protein. Serial dilutions of certain experimental conditions. Recent studies have demonstrated
biological samples can be used to identify the appropriate linear range regulated expression of actin, tubulin, and other HKPs in response to
of sample loading for both the target protein(s) and internal loading conditions such as cell confluence, disease state, developmental stage,
control. It is important to note that this linear range will likely be dif- hormonal state, drug treatment, and cell or tissue type
ferent for each target protein and loading control combinations and [33,35,37,41–49]. Manceau et al. [43], demonstrate the variability in
may be affected by the detection chemistry and/or imaging platform. β-tubulin and β-actin across different tissues extracts from mouse
The relationship between sample input and band intensity must be both neonates. Because the variability of HKP expression in experimental
linear and proportional for both target protein and internal loading samples will introduce error in quantitative analysis, validation is an
control to enable quantitative analysis. Choosing an appropriate ILC important first step. Before an HKP or other single-protein loading
may seem simple, but it is an important and sometimes obscure aspect control is used for quantitative analysis of Western blot data, its stable
of experimental design for accurate quantitative analysis of Western expression should be demonstrated and verified for the relevant ex-
blot data [6,10]. perimental samples and manipulations [3,29,33–35,37,47].
2.3.1. Internal loading controls 2.5. Internal loading control: total cellular protein
The ILC must meet the two requirements when evaluated in the
specific conditions, treatments, cell or tissue types, and experimental In recent years, total cellular protein staining of the blotted mem-
context where it will be used. Because experimental manipulations may brane has emerged as the preferred method for normalization of sample
influence the expression of the ILC, biological stability should be vali- loading in quantitative analysis of Western blot data
dated in all samples that represent the intended conditions and treat- [35,37,41,44,47,50–53]. Prior to the immunodetection of the blot, a
ments. Biological variability in expression of the ILC is a source of error fluorescent total protein stain is used to visualize and assess actual
that may undermine the accuracy of the analysis; particularly for ana- sample loading across the blot. The membrane is then processed for
lysis of small differences in target abundance [3,11,33–35]. The antibody incubations and detection. Total protein loading controls are
variability introduced by technical limitations (gel matrix, transfer much more resistant to biological variability than an HKP or other
conditions, buffers, etc.) of the Western blot method should also be single-protein loading control [3,15,31,35,44,47,50,51] and may pro-
considered when choosing and validating an ILC. Either type of varia- vide a wider linear range of detection than an HKP – and because of its
bility may make it more difficult to reliably detect small differences and resistance to biological variability, it requires less validation prior to
effects, leading to false-negative results [4,15,36,37]. use [3,4,7,15,35,41,44,50,51]. Because variation is often introduced
Normalization of a target protein is most accurate when the target during electrophoretic transfer from the gel to a membrane, total pro-
protein and ILC are detected in the same lane on a single blot tein staining of the blot is preferable to staining of the gel prior to
[6,8,38,39]. To achieve this goal, detection must be performed in the transfer or staining of a duplicate gel loaded with the same samples
combined linear range – the range of sample loading that produces a [3,35,47,51]. Any method used for the detection of total protein
linear, proportional response in band intensity for both the target and loading should not damage the bound sample proteins or interfere with
the ILC [3,6,7,29,33]. If they cannot be accurately detected in the same subsequent antibody-based detection of the blot [6,31,35,54,55]. A
range of sample loading, the results will not be meaningful; a different total protein stain should meet a number of criteria, including an ap-
ILC should be selected for the experiment. This may occur for a variety propriate linear range of detection that is compatible with the target of
of reasons, such as the use of an abundant HKP as an ILC for a low- interest; stable signals and low sample-to-sample variability; lack of
abundance target, a pan/PTM analysis experiment where an abundance interference with immunodetection and other downstream analysis;
of unmodified and modified forms varies dramatically, if primary an- suitability for membrane staining, to correct for variability during
tibody specificity and affinity are poor, or if transfer methods require transfer; and compatibility with multiplex detection and quantification
further optimization. [3,6,35,41,47,51–53,55,56]. Several fluorescent membrane stains have
recently been described in the literature, including Ponceau Stain [55],
2.4. Internal loading control: housekeeping protein Coomassie Brilliant Blue, SYPRO Ruby stain, Blot FastStain [57], and
Revert 700 Total Protein Stain [35,58–62].
A single endogenous reference protein, such as a housekeeping
protein, is often used as an ILC. Members of Actin, tubulin families and 2.6. Detection chemistry
glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) are common ex-
amples. This method is referred to in the literature as a single-protein Two main types of detection chemistry are used to visualize signals
loading control [3]. Although this method is widely used and can be on Western blots. Enhanced chemiluminescence (ECL) is a popular
quite accurate, it is uniquely vulnerable to biological variability [40]. enzymatic method that uses horseradish peroxidase (HRP) as an in-
An HKP or other single-protein loading control alters and reformulates direct reporter of secondary antibody binding. This HRP reporter pro-
the experimental hypothesis, as described by Aldridge et al. [3]. Rather duces photons of light as it consumes a luminol-based substrate.
than evaluating the abundance of the target protein relative to total Fluorescence detection is a non-enzymatic method that uses fluor-
sample protein or cell number, the experiment now examines target ophore-labeled secondary antibodies as a direct reporter of antibody
abundance relative to that particular single-protein loading control [3]. binding. Although chemiluminescence is widely used, its inherent en-
If the expression of that single protein is stable and unaffected by the zyme/substrate kinetics are a source of variability and often fail to
relevant experimental conditions and samples, this ILC meets the first produce the linear, proportional signal response required for quantita-
requirement and the hypothesis is valid. But if the expression of that tive analysis of Western blot data [5,6,8,11,63]. The rate of the che-
single-protein loading control is variable, the reformulated hypothesis miluminescent reaction is dependent on the local concentration of en-
will fail. zyme and substrate. Substrate availability is a dynamic property, and
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
the reaction rate will vary continuously over time and across the surface 2.7. Normalized Western blot data
of the blot. A low-intensity band will consume the available substrate
much more slowly than a strong band with a high local concentration of Normalization mathematically corrects for small, unavoidable var-
HRP reporter that may rapidly deplete the available substrate. If the iations in sample loading and protein transfer by comparing the target
HRP concentration is too high, a signal may be lost in areas where the protein to the ILC in each lane. The target protein signal in each lane is
substrate is rapidly consumed and exhausted; this is the cause of ghost divided (normalized) by the signal value for the ILC in that lane. When
bands (reverse banding) and “burned-in” bands with brown or yellow target signals are normalized to sample loading, relative levels of the
precipitate [6,64]. Different chemiluminescent substrate formulation target protein can be compared across the blot to determine if changes
can be specifically used to detect femtograms (ultrasensitive), pico- in band intensity represent biological differences between samples.
grams or milligrams of target protein within a lysate [65,66]. As such Normalization is based on the fundamental assumption that both the
simultaneous detection of high and low concentration of proteins in the target and ILC signals are dependent on sample concentration. For this
same blot can be challenging with one formulation ECL substrate assumption to hold true, as mentioned above the ILC must meet two
[67–69] thereby, resulting in a non-linear and narrow dynamic range of requirements: it must be expressed at a generally constant level across
detection. However, titration of protein amount, the primary antibody all relevant experimental samples and conditions; and the resulting
can help in mitigating all the above-mentioned issues, provided the signal intensity must be proportional to abundance, without saturation
scientist is cognizant [52,70]. effects or technical limitations. A loading control that does not meet
We and others developed fluorescent Western blot methods as a both requirements is not an accurate indicator of sample loading and
more convenient and quantitative alternative to indirect chemilumi- should not be used for normalization and quantitative analysis of
nescence [63,71]. Fluorophores are retained at the site of antibody Western blot data [3,31,47,51].
binding and emit photons upon exposure to the appropriate wavelength Without normalization, an apparent difference in target abundance
of excitation light, typically in the near-infrared (NIR) spectrum on a Western blot cannot be accurately interpreted. This concept is il-
[5,6,8,38,71–73]. Signals are very stable and much more reproducible lustrated in Fig. 4. Detection of the target protein is seen in Fig. 4A
because they are unaffected by timing, substrate availability, and other (green). Plotting the raw intensity values for the target may suggest that
variables that limit the usefulness of chemiluminescent methods for target abundance is variable among the different samples (Fig. 4C).
quantitative analysis [5,6,8,11,74]. Fluorescence imaging also enables However, the observed difference between target bands may be the
multiplex detection of a target protein and ILC in the same lane on the result of inconsistent loading of total protein extract (Fig. 4B). Sample
same blot, for more accurate correction of sample-to-sample and lane- #4 appears to have reduced levels of target protein compared to rest of
to-lane variation [6,8,38,63]. Stripping of Western blots for re-probing the samples, however, after normalization to correct variations in
can cause substantial loss of sample proteins from the membrane and is sample loading; it is evident that sample extracts were loaded unequally
an avoidable source of error [6,8,30,31]. Fluorescence is generally re- in each lane and therefore, any changes in target protein observed may
cognized as the most accurate and reliable method for quantitative be due to improper loading of protein extracts and not due to experi-
analysis of Western blot data [4–8,41,73,74]. Source of species in which mental intervention (Fig. 4D).
antibodies were raised, the wavelength of secondary antibody tagged Normalization is appropriate for the correction of small and un-
fluorophore, and imaging system needs are critical to performing avoidable differences in cell number, sample concentration, loading
fluorescence-based Western blot detection of proteins. error, and position or edge effects from electrophoresis and transfer.
Saturation artifacts are not readily apparent in a Western blot image However, it should not be relied on to correct for preventable error and
and may go undetected if serial dilutions of a sample are not examined variability in the quantitative analysis of the Western blot process [3,6].
to define the linear range of detection. Two types of saturation are Normalization should be used in addition to careful experimental de-
frequently observed in quantitative analysis of Western blot data. sign and sample preparation, not in place of it. Minimizing or elim-
Membrane saturation is caused by the overloading of the sample inating sources of variation throughout the experiment is critical for
protein. During the transfer of an overloaded gel to a blotting mem- reproducible quantitative analysis of Western blot data (see Refs.
brane, abundant sample proteins bind in layers on the surface of the [6,7,29,78] for excellent information about protein extraction, sample
membrane [11,15,31,76]. If highly abundant proteins exceed the local handling, and other error-prone aspects of the Western blot method).
binding capacity of the membrane, they will be washed away. The Total protein normalization is an aggregate method that combines
layering of abundant proteins also limits antibody access and may only the band intensities from many different sample proteins in each lane –
allow antibodies to bind the top layer of protein. This layering effect essentially using multiple ILC proteins to provide a more accurate
causes underestimation of strong bands and may also interfere with the readout of sample loading. This multi-protein approach is also called
detection of low-abundance proteins on the blot. Membrane saturation normalization by sum [36]. This normalization method also offers a
is common may be difficult to identify without the evaluation of a di- more direct readout of the cellular material loaded than antibody-based
lution series of the sample. This type of saturation can affect any detection of a single endogenous protein [3,4,31,35,36,41,44]. Total
Western blot assay, regardless of the detection chemistry or imaging protein normalization is now increasingly viewed as the “gold stan-
method [11,15,77]. dard” for performing quantitative analysis of Western blot data [52,53].
Signal saturation occurs when the intensity of a strong band ex- The Journal of Biological Chemistry recommends normalization to total
ceeds the capacity of the detection chemistry or imaging system. At this protein loading, clearly stating their preference “that signal intensities
point, strong signals begin to plateau and increasing amounts of target are normalized to total protein by staining membranes with Coomassie
no longer produce the expected increase in signal (Fig. 3). Chemilu- Blue, Ponceau S, or other protein stains” [52,53].
minescent detection is highly susceptible to saturation; the generation
of a signal is restricted by substrate availability, and the linear range is 3. Background subtraction
quite limited, even when detected by digital imaging [5–7,11,15,74].
Fluorescent Western blot methods are relatively resistant to saturation Western blots not only contain the signal generated by the im-
and provide a much broader linear range, particularly when imaged munodetection of the target protein but also signal generated as a result
with a digital system that provides an appropriately wide dynamic of nonspecific bands, smears, and the inherent signal produced by the
range [5–8,73,74]. However, in heavily loaded samples, self-quenching membrane itself. When a target band is quantified the background
of tightly packed fluorophores may be possible [6]. signal around the band is also included in the data analysis, and the
goal is to reduce/remove the confounding error introduced by such
background signal [23,79,80]. Currently, several Western blot analysis
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
Fig. 3. Saturation of strong bands obscures actual differences between samples. Increasing amounts of C32 (sc-2205) whole cell lysate (1–60 μgs) were loaded
on 4–12% Bis-Tris PAGE gels and transferred onto a PVDF membrane. Fluorescent detection of Actin was detected on three replicate blots performed on different
days. A) Actin bands were analyzed and quantified with Image Studio™ analysis software (LI-COR Biosciences) and the detected band intensities compared to the
predicted signal intensity, based on the known amount of sample protein loaded in each lane. Results of three replicate experiments are shown (blue line indicates
mean values; dotted line shows predicted band intensity for a linear and proportional response. Above 10 μg of cell lysate per lane, actin signals begin to plateau, and
increased sample loading does not produce the expected increase in band intensity. B) Differences between high-intensity bands were underestimated by quantitative
analysis. Pink bars show the actual mean band intensity of replicate blots [ ± Standard Deviation (STDEV.)]; blue bars indicate the predicted increase in band
intensity. A comparison of the 5 μg and 20 μg sample lanes indicates a 3.1-fold increase in signal, lower than the predicted 4-fold increase. Comparison of the 10 μg
and 30 μg sample lanes indicates a larger discrepancy in band intensity: a 1.6-fold increase is observed, roughly half of the expected 3-fold change. At or above 30 μg
of sample, increases in band intensity are minimal and differences between lanes are not reproducibly detected (saturated). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
software packages offer multiple options to subtract background signal works well with real-life Western blots that have oddly-shaped mis-
and quantify target bands. Each Western blot has a unique profile with shapen bands and smears, and the data generated has greater accuracy
respect to artifacts, uniformity of background, positioning of lanes and than local and global subtraction methods (For more details read [85];
bands and requires a background subtraction algorithm that can adapt Fig. 5).
to the said variations.
There are several methods to subtract the background from the 4. A systematic approach to accurate quantitative analysis of
Western Blot image [23,79,81–84] either using the Shape-Based Western blot data
methods for local and/or global background subtraction; Lane-Based
methods such as Rolling Ball subtraction. A new patent-pending Here, we describe a five-step systematic strategy to increase the
method called Adaptive Background Subtraction (ABS) removes user- precision and reliability of the quantitative data generated from the
bias by taking into consideration both shape as well as lane profiles to Western blotting technique. This process incorporates critical valida-
generate reproducible and accurate quantitative Western Blot data. ABS tion steps that address common, but sometimes unrecognized, sources
background subtraction algorithm present in Empiria Studio Software of error and variation. Because we and others strongly prefer and
Fig. 4. Normalization enables accurate interpretation of differences in protein expression. This is a simulated Western blot representing unequal loading of
sample extracts to visualize a target protein and total protein extract via antibody and Total Protein Stain respectively. Total protein will be used as an ILC in the
simulated data analysis. A) This illustration depicts Western blot results for a target protein in lanes 1–5 (green bands). Lanes 3 and 4 appear to have higher signal for
the target protein compared to lane 4. B) The same blot from panel A is presented to depict the total protein extract in lanes 1 through 5 (red). Lanes 3 and 4 appear to
have higher total protein extract compared to lane 4. C) Raw intensity values for the target protein bands plotted against the sample number suggest varying
expression levels of the target protein. D) Upon normalization of the target protein bands in panel A with the total protein signal in panel B it is evident that sample
extracts were loaded unequally in each lane and therefore, any changes in target protein observed may be due to improper loading of protein extracts and not due to
experimental intervention. Such a Western blot would not provide accurate and reproducible qualitative data. R.F.U; Relative fluorescence units. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
Fig. 5. Quantitative analysis with Adaptive Background Subtraction (ABS) is more reproducible than local and global background subtraction. Multiple
users analyzed the designated bands on each blot (arrowheads on images) using local, global/user-defined, and Empiria Studio ABS background subtraction.
Variability of background-corrected band intensity, expressed as the mean % CV of the quantitative results for that image, was compared for each background
subtraction method. A) Blot A displayed oddly shaped bands and smeared trails in sample lanes. Graph shows the mean CV of quantitative analysis for each
background method (error bars show standard deviation, n = 6). B) On Blot B, faint bands were surrounded by background in sample lanes. Graph shows the mean
CV for each method (error bars show standard deviation, n = 6). C) The designated bands on Blot C were smeared and misshapen. Graph shows the mean CV for each
method (error bars show standard deviation, n = 5).
recommend fluorescent Western blot methods for quantitative analysis preference to bind the target antigen in the presence of a heterogeneous
[5–8,52,63,74], this strategy was specifically developed for use with mixture of sample proteins. These characteristics should be verified and
fluorescence-based detection. validated for each primary antibody. Because antibody performance is
greatly influenced by the assay context and parameters, validation
4.1. Validate the primary antibody should be performed by the user in the intended assay and relevant
experimental context [18,86–94]. The reproducibility of results pro-
After sample proteins are separated on a protein gel and transferred duced with the primary antibody should also be verified [18,76,88,95].
to the membrane, primary and secondary antibodies are used to bind
and visualized the target protein. This process relies on two key prop- 4.1.1. Specificity
erties of the primary antibody: specificity, the antibody's ability to re- The specificity of antibody within the realm of a Western blot means
cognize and bind to the target antigen; and selectivity, the antibody's that the antibody recognizes the target protein(s), either as a single
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
Fig. 7. Identification and validation of an appropriate HKP in the MAPK/ERK pathway. Cell extracts prepared from untreated (UT) or Epidermal growth factor
(EGF) (ET) treated A431 cells were loaded onto 10% 10% Bis-Tris gel (NP0303BOX) and run under the MOPS buffer system. All the blots were incubated with
Intercept Blocking Buffer for one hour at room temperature. Technical replicates for both UT and ET categories are presented. Proteins belonging to MAPK/ERK
pathway were selected to be tested via Western blot method. Total protein extract was visualized in each blot (a, a’, a”) using Revert 700 Total Protein Stain (LI-COR
P/N 926-11010). Blots; b, b’, and b” were incubated with Anti-Ras antibody (CST #3965; Lot #1); Anti-CDK4 (D9G3E) antibody (CST #12790; Lot#14); and Anti-
CDK6 (DCS83) antibody (CST #3136; Lot #4). Merged images of the blots (c, c’, c”) imaged under different acquisition channels; 700 nm for Revert 700 (a, a’, a”) and
800 nm for Ras, CDK4, and CDK6 (b, b’, b”) highlight uniform loading of sample in each lane. Ras, CDK4, and CDK6 levels appear to be unchanged between untreated
and EGF treated A431 cell extracts (d, d’, d”). F test to compare variance, P value > 0.05). Sample loaded: 5 micrograms; Blocking buffer: Intercept Blocking Buffer
(TBS); Intercept T20 (TBS) Antibody Diluent; Protein ladder: Chameleon duo ladder (LI-COR P/N 928-70000; Lot# C70803-03). Imager: Odyssey® CLx; resolution:
169 μm; Intensity: auto mode.
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
Fig. 8. Determination of the combined linear range of detection for CPARP, β-ACTIN (ACTB), GAPDH, and COXIV. Jurkat cell extracts harvested from cells
treated with 25 μM Etoposide for 5 h at 37 °C was loaded on 4–12% Bis-Tris NuPage gels and electrophoresed under the MOPS buffer system. The blots were blocked
with Odyssey Blocking buffer- TBS for one hour at room temperature and subsequently incubated with primary antibodies against CPARP (CST #5625; Lot#13),
ACTB (LI-COR P/N 926-42210; Lot #C80322-01), GAPDH (CST #5174; Lot #2), and COXIV (LI-COR P/N 926-42214; Lot #C5033324-02) (~1 μg/ml) at 4 °C
overnight with gentle shaking. Subsequently, the primary antibody binding was visualized by using IRDye 800CW Goat anti-Rabbit IgG (H + L). Sample loaded:
1.25–40 micrograms; Blocking buffer: Odyssey Blocking Buffer (TBS); Protein ladder: Chameleon duo ladder (LI-COR P/N 928-70000; Lot# C70803-03). Imager:
Odyssey® CLx; resolution: 169 μm; Intensity: auto mode. Linear range of detection for all the proteins are plotted in (A). Linear range of CPARP and COXIV (B)
appears to have overlapping range of detection compared to CPARP and ACTB (C) and CPARP and GAPDH (D).
to be used as a valid indicator or proxy of sample loading protein loaded in each lane (Fig. 7a, a’, a”). No significant changes in
[3,29,33–35,37]. Stability should be demonstrated before an HKP or protein expression were observed for Ras, CDK4, and CDK6 proteins
other single protein is used for quantitative analysis of Western blot (Fig. 7b, b’, b”; d, d’, d”), and therefore, is suitable to be used as HKP for
data normalization, to ensure that expression of the ILC is not affected the samples under consideration.
by experimental treatments or manipulations [29,33,34,47,53,111].
4.3. Two-sides of the same coin: Post-Translationally modified (PTM)
4.2.1. Stable or Unstable: ILC? proteins
Expression of the HKP or other single-protein control should be
carefully examined in samples that represent the desired experimental Western blots generated to detect PTM proteins utilize an ILC that
conditions. A comparison of HKP levels to total protein loading in these accounts for the total presence of the unchanged protein (also known as
samples is a straightforward way to demonstrate the stability of ex- Pan-protein). Here, a modification-specific primary antibody is multi-
pression. Alternatively, HKP levels may be compared to an unrelated, plexed with a pan-specific primary antibody that recognizes the target
endogenous protein already shown to be stably expressed in the re- protein in any modification state [6,38,71,72,112–114]. The pan- and
levant experimental samples and conditions. If changes are made in modification-specific antibodies should be derived from different host
experimental treatments, cell line, tissue type, cell density, or other species, so they can be discriminated by secondary antibodies labeled
relevant experimental parameters, stable HKP expression, as well as a with spectrally distinct fluorophores [63]. Pan/Phospho and other
linear range of detection, must be re-validated for the new conditions types of pan/PTM analysis are widely used to monitor and compare
[33,34,47]. Several proteins belonging to the MAPK pathway were relative changes in protein modification across a group of samples. Pan/
tested for stability in both vehicles treated and etoposide treated Jurkat Phospho analysis is specifically recommended by the Journal of Biolo-
cell extracts (Fig. 7c, c’, c”). Each blot was also stained to visualize total gical Chemistry, which stipulates that “phospho-specific antibody signals
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
Fig. 9. Combined Linear Range (CLR) detection of Total protein extract and OCT-4 in HEK293T cells. 4-fold dilution of cell extracts from HEK293T untreated
control (a’-a”) as well as knockdown HEK293T (experimental) (b-b”) category were loaded onto 10% Bis-Tris gel (NP0303BOX) and run under the MOPS buffer
system. All the blots were incubated with Revert 700 Total Protein Stain (LI-COR P/N 926-11010) to visualize total protein extract in each lane. Subsequently, the
blots were incubated in Intercept Blocking Buffer for one hour at room temperature and thereafter in Anti-Oct-4 antibody (CST #2750; Lot #4) and IRDye 800CW
Goat anti-Rabbit IgG (H + L). (a-a”) Individual linear range of detection for total protein and OCT-4 was determined for control cell extracts (a - a”) as well as for
experimental cell extracts (b – b”). Combined linear range for total protein extract and OCT-4 protein detection was plotted for both cell extract types (c, d). CLR was
identified at 4.5 micrograms for both cell types. Blocking buffer: Intercept Blocking Buffer (TBS); Intercept T20 (TBS) Antibody Diluent; Protein ladder: Chameleon
duo ladder (LI-COR P/N 928-70000; Lot# C70803-03). Imager: Odyssey® CLx; resolution: 169 μm; Intensity: auto mode.
should be normalized to total levels of the target protein” [97,111]. 4.4. Define the linear range of detection
Although pan/phospho analysis is the most commonly performed type
of pan/PTM analysis, other protein modifications are also studied with Using the validated antibodies, it is important to determine the
this method (ubiquitination and palmitoylation analysis). appropriate amount of sample to load to generate data that can be
Pan-protein normalization accounts for changes in expression of the quantified. Normalization and analysis must be performed in the linear
target protein, which might confound analysis of the abundance of the range of detection, where a linear and proportional response is ob-
modified form. It also eliminates the stripping and reprobing of blots, served between sample loading and band intensity.
which introduces error by causing loss of blotted proteins from the
membrane [6,31,38]. Because the unmodified and modified target 4.4.1. Finding the combined linear range for a target protein and internal
protein is detected on the same blot and in the same lane, normalization loading control
can correct for transfer artifacts in the blot. Pan/PTM analysis should Careful examination of the linear range is particularly important for
only be used for comparison of relative abundance; it does not provide HKPs and other single-protein loading controls. The strong bands pro-
information about the stoichiometry of the modification [77]. duced by highly abundant ILC are frequently affected by saturation. If
the abundance of a target is substantially lower than the single-protein
loading control, the combined linear range may be very narrow. Total
4.3.1. Publication guidelines protein normalization typically provides a wider combined linear
If an HKP is used for quantitative analysis of Western blot data range, without the rapid saturation observed for HKPs
normalization, authors should be prepared to provide evidence that its [35,44,50,51,55,56]. This wider combined linear range is very helpful
expression was not affected by the experimental treatments applied for low-abundance target proteins, which often require the loading of
[53,111]. The editors of the Journal of Biological Chemistry have ex- large amounts of sample protein and cannot be used with a highly
pressed a preference for total protein normalization in quantitative abundant HKP.
analysis of Western blot data [53]. The experiment in Fig. 8 demonstrates the differences in a linear
range that can be observed between different HKPs. Cleaved PARP (c-
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L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
PARP) and three different HKPs (β-actin, COX IV, and GAPDH) were normalized signalExperimental sample
Fold change =
detected in lysates from Jurkat cells treated with 25 μM Etoposide for normalized signalControl sample
5 h at 37 °C (check material and method section for more details).
Because the target and HKPs span a range of MW and are well resolved A ratiometric analysis is often performed to enable a relative com-
by electrophoresis, all four proteins could be detected simultaneously parison of target abundance across a group of samples. The normalized
on a single Western blot. This facilitates comparison by eliminating target signal for each sample is divided by the normalized target signal
blot-to-blot inconsistencies in sample loading, electrophoresis, and observed in the control sample. These ratios express the abundance of
transfer. Fig. 8A shows a combined linear range for poly (ADP-ribose) the target protein as a fold or percentage change, relative to the control.
polymerase cleavage (CPARP), GAPDH, COXIV, and β-actin (ACTB). In A comparison of all samples to the experimental control produces re-
Fig. 8B, the combined linear range for CPARP and COX IV is linear lative values that are unitless, proportional, and independent of the raw
between ~7.5 and 30 micrograms. However, there is no useable com- signal intensity. Fold change values > 1.0 indicate increased abun-
bined linear range for CPARP, ACTB, and GAPDH (Fig. 8C and D). In dance relative to the control, and values < 1.0 denote decreased
these samples, band intensity for GAPDH and ACTB begins to saturate abundance. Percentage change expresses the same information as a
and plateau at ~15 μg of sample protein loading. percentage, with a positive value indicating increased relative abun-
Working in the middle of the combined linear range helps to limit dance and a negative percentage indicating decreased abundance
error and variability introduced by high- and low-intensity data points. (Table 1). Fig. 10 demonstrates the normalization of CPARP protein to
With an HKP or other single-protein loading control, high-intensity data Total protein extracts from Jurkat cells exposed to three different ex-
points introduce error and should not be used for normalization; these perimental conditions. Untreated, 33% spiked Etoposide lysate, and
data points will increase the mean Coefficient of Variance (CV) of the 100% Etoposide treated lysates are loaded onto a PAGE gel in technical
normalized data and should be avoided [15,36]. From a statistical replicates (n = 3). Signal values for total protein, CPARP abundance
perspective, variability introduced by a single-protein loading control from each category of cell extracts are represented in 10A′-B’. Fig. 10C’
may increase the frequency of false-negative results (small, but statis- reveals the fold change in expression of CPARP in 33% (~2 fold) and
tically significant, differences between samples that are not identified 100% (~3.3 fold) Etoposide exposed extracts.
during data analysis).
If validation experiments fail to identify a combined linear range of 4.5.1. Publication guidelines
detection, as in Fig. 8C–D, a different ILC may be required. The use of Methods used to quantify and normalize signal intensities should be
total protein for normalization decreases the variability of high-in- disclosed, along with data analysis methods and software tools used for
tensity data points but does have the potential to increase variation for quantification and analysis. Raw data showing image analysis and
low-intensity measurements [36]. Fig. 9 demonstrates the combined quantification, including original digital images of Western blots, may
linear ranges for OCT-4 protein in HEK293T cell extracts derived from be requested during peer review [13,53,111,115].
control and experimental categories. For both lysates, 4.5 micrograms
appear to be in the middle of the combined linear range of detection for 4.6. Replicate and reproduce
Total protein extracts and OCT-4 protein. Optimization of transfer
methods, sample loading, antibody choice, detection chemistry, or Replication of quantitative analysis of Western blot data results
other assay parameters may help to improve the linear range of de- confirms the validity and reproducibility of any observed changes
tection for the selected ILC. In general, increasing the number of in- [13,14,18,20,21]. Replicate measurements help to ensure that the ex-
ternal control proteins will reduce the mean CV and improve the ac- perimental effects we report are reproducible – that they represent
curacy of normalization, making it easier to reliably detect small or actual differences between samples, rather than artifacts of experi-
subtle differences between samples [3,6,35,36,51]. mental variability or noise [12,18]. Replication is essential when ana-
lyzing small or subtle differences between samples, to characterize and
understand the contribution of error and the limits of quantitative
4.4.2. Publication guidelines analysis [12,19,20]. Two types of replicates, technical and biological,
When quantitative analysis of Western blot data results are re- are commonly used to address different questions in quantitative ana-
ported, authors should disclose how signal intensity was quantified as lysis of Western blot data.
well as how the linear relationship between sample loading and signal
intensity was confirmed for each antigen [53,111]. 4.6.1. Technical and Biological Replicates
Technical replicates are repeated measurements of the same sample,
used to characterize the precision and variability of any assay or
4.5. Data analysis method [19,20,116]. Common examples include loading the same
sample in multiple lanes on a Western blot, running replicate blots in
Data analysis of Western blot data sets should be performed on parallel, or running the same kind of gel multiple times on different
biological as well as technical replicates generated using the validated days. High variability between technical replicates makes it more dif-
antibodies, combined linear range of detection, and valid ILC estab- ficult to separate an observed experimental effect from the inherent
lished in the prior steps. After quantification of target and ILC signals, variation of the Q WB assay [12,19,20,53,117]. As a result, small but
data analysis is performed. genuine differences between samples may not be detected.
Analysis typically begins with the designation of the experimental Biological replicates are parallel measurements of independent and
control sample that will be used for relative comparison. The lane biologically distinct samples that are intended to control for random
normalization factor is then calculated for each lane, using the HKP biological variation [9,19–21]. A biologically relevant effect should be
band intensity values or combined total protein signal values for each observed reproducibly in independent samples. Analysis of samples
lane (Fig. 10A). This factor is calculated by dividing the signal intensity derived from multiple individuals or from multiple, independent cell
value for each experimental sample by the signal intensity observed for cultures are common examples. In some cases, a similar experimental
the control. To apply the normalization factors, the signal intensity of effect may be demonstrated in a different biological system or context
the target band in each lane is divided by the lane normalization factor [13,20,86,117]. Biological variability is generally expected to be higher
for that lane. This process generates the normalized signal intensity than technical variability [12,16,20]. Considerations for choosing ap-
value for each sample. propriate replicates are described by others [19–21]. The replication
strategy should be developed prior to the generation of quantitative
11
L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
12
L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
different conclusions than the full dataset [118,119]. Weissgerber et al. 6. Materials and methods
[119] recently introduced free, online interactive tools designed to
improve data visualization for small datasets. The “interactive dot plot” 6.1. Cell culture
tool allows different graphs to be viewed as univariate scatterplots, box
plots, and violin plots. It also enables visualization of clustered non- A431 (ATCC® CRL-1555™), Jurkat (ATCC® TIB-152™) were cultured
independent data, such as technical replicates. The “interactive re- at 37 °C in a humidified atmosphere containing 5% CO2 as per ATCC
peated experiments” is designed for visualization and comparison of guidelines. Cells were cultured up to 80% confluence before being
data from repeated, independent experiments. treated or harvested to be stored at −80 °C.
13
L. Pillai-Kastoori, et al. Analytical Biochemistry 593 (2020) 113608
Author contributions and is it significant? A brief discussion of statistics and experimental design, EMBO
Rep. 13 (2012) 291–296.
[23] J.J. Bass, D.J. Wilkinson, D. Rankin, B.E. Phillips, N.J. Szewczyk, K. Smith,
A.R.S.G co-wrote the main manuscript text and prepared Figures. P.J. Atherton, An overview of technical considerations for Western blotting ap-
J.A.H supervised, reviewed, and proof-read the manuscript. LPK de- plications to physiological research, Scand. J. Med. Sci. Sports 27 (2017) 4–25.
signed and conducted the experiments, wrote the main manuscript text [24] A. Pandey, K. Shin, R.E. Patterson, X.-Q. Liu, J.K. Rainey, Current strategies for
protein production and purification enabling membrane protein structural
and finalized the Figures. ARSG and LPK are co-first authors of this biology, Biochem. Cell. Biol. 94 (2016) 507–527.
manuscript. [25] P.T. Wingfield, Overview of the purification of recombinant proteins, Curr. Protein
Pept. Sci. 80 (2015) 6.1.1-6.1.35.
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Declaration of competing interest importance of Lysis buffer choice, in: B.T. Kurien, R.H. Scofield (Eds.), Western
Blotting: Methods and Protocols, Springer New York, New York, NY, 2015, pp.
L. Pillai-Kastoori, A. Schutz-Geschwender, and Jeff A. Harford are 49–60.
[27] K. Gavini, K. Parameshwaran, Western Blot (Protein Immunoblot), StatPearls,
employees of LI-COR Biosciences.
(2019) Treasure Island (FL).
[28] E.I. Miskiewicz, D.J. MacPhee, Lysis buffer choices are key considerations to en-
Acknowledgments sure effective sample solubilization for protein electrophoresis, in: B.T. Kurien,
R.H. Scofield (Eds.), Electrophoretic Separation of Proteins: Methods and
Protocols, Springer New York, New York, NY, 2019, pp. 61–72.
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