REPRINT FROM

Biotechnology from bench to business

Volume 31, Number 20 November 15, 2011

OMICS

Bioprocessing

Drug Discovery

Clinical Research & Diagnostics

Biobusiness

Drug Discovery Assay Tutorial

Stain-Free Approach for Western Blotting
Alternative to the Standard
Blot Normalization Process
Ryan Short and Anton Posch, Ph.D.

W

estern blotting is one of themost ubiquitous procedures
used in the life science research lab today. Commonly used toassess relative protein expression differences, accurate Western blot quantitation
requires careful attention to procedural
details and normalization.
The most common normalization
method is the use of housekeeping proteins (HKPs). This article will review thefrequent challenges of using HKPs fornormalization and propose an alternate
method for verifying HKP results, and/or possibly replacing HKP normalization.
Tubulin, GAPDH, and actin are frequently chosen as HKPs due to their general lack of variability associated withchanges in experimental conditions. As
a number of publications have shown,however, caution should be used whenselecting HKPs since not all HKPs areconstant under changes in experimental
conditions or sample types. For this rea-A

Figure 1. (A) HeLa cell lysate (2.5–80 µg total protein per lane) separated
by TGX “any kD” Criterion Stain-Free 1-D SDS gel (18 slots) and imaged
with ChemiDoc MP. (B) HeLa cell lysate (2.5–80 µg total protein person, it is often recommended that results
are validated using multiple HKPs, adding time and complexity to Western blot
experiments.
The process of using HKPs for blot
normalization can be daunting. Two widespread techniques are “strip and re-probe”
and multiplex fluorescent detection.
HKP Normalization Methods
Using the strip and re-probe method,
the protein of interest is probed and detected. Antibodies and detection chem-Blane) separated by TGX “any kD” 18 slot Criterion Stain-Free 1-D SDS
gels, blotted to nitrocellulose membrane and imaged with ChemiDocMP. M = marker proteins.

Figure 2a. LCL cell lysates (30 µg total protein per lane) separated
by TGX and kD Criterion Stain-Free 1-D SDS gel, blotted to PVDF
membrane and imaged with ChemiDoc MP.

AB

Figure 2b. Immunofluorescence imaging of DNA replication factor
MCM7 and housekeeping protein GAPDH (serves as loading control) ofLCL samples with ChemiDoc MP at different wavelengths. Monoclonal
antibodies against MCM7 (mouse) and GAPDH (rabbit) were diluted
1:1,000 and 1:2,500 respectively. Secondary antibodies from Rockland
were antirabbit DyLight 549 (1:10,000) and antimouse DyLight 649(1:20,000).

istries are then stripped from the membrane using some combination of heat,
detergent, or reducing agent. The blot
may then be re-probed with HKP specificantibody and re-detected. Not only is theprocess time consuming, but inevitably,
the stripping process will remove somelevel of antigen, thereby compromising
downstream results.
Multiplex fluorescent Western
blotting is a more elegant solution,
whereby multiple antigens can be simultaneously probed and detected using multiple fluorescently labeled secondary antibodies. Western blotting
frequently requires optimization ofblocking reagents, antibody concentrations, and incubation times. Users need
to be mindful of challenges like antibody cross-reactivity, and should havean optimization process in place thatvalidates the detection of each antigen
separately, before attempting a multiplex detection.
The challenges associated with the useof HKPs by stripping and re-probing, oroptimization of multiplex fluorescent blot
detection, can be avoided. A new, easier
method uses Stain-Free technology for
total protein Western blot normalization.
Benefits of Stain-Free Technology
Stain-Free technology is a uniquein-gel chemistry that is available in BioRad TGX Stain-Free precast gels. The
gel formulation incorporates a trihaloNovember 15, 2011|

compound that when exposed to UV
irradiation activates a covalent reaction
between the trihalocompound and tryptophan residues on the proteins in thegel, resulting in UV induced fluorescence.
Stain-Free technology enables fluorescent visualization of 1-D SDS PAGE
gels and corresponding blots using BioRad Laboratories’ (www.biorad.com)
ChemiDoc MP imaging system (Figure1A). The quality of SDS-PAGE separations before blotting can be easily monitored and the transfer efficiency of theblotting process can be quickly inspectedby imaging both the membrane (Figure1B) and the SDS gel after the blottingprocess. Furthermore, using Image Lab
4.0 software, which is included with theChemiDoc MP, the relative amount of
total protein in each lane on the blot canbe calculated and used for quantitation
normalization.
Stain-Free technology offers comparable sensitivity to conventional blot stains
such as SYPRO Ruby and Ponceau S,and provides better reproducibility andlinearity. Linear range of the Stain-Freetechnology is up to 80 µg protein for 18well and up to 110 µg per lane for 12well Criterion mid-size gels. This range
fits well with usual protein loads in quantitative Western blotting experiments andenables loading control calculations overa wide protein-loading range.
HKP-based normalization with GAPDH, actin, or tubulin needs to be opti-genengnews.com|

mized for antibody dilutions, incubation
times, and imaging settings. This is a very
lengthy process compared to Stain-Freetechnology, which gives accurate, standardized protein-loading control with nooptimization.
Protein-Profiling Studies
Proteomic technologies like 2-D PAGE
are customarily used in semi-quantitative
protein-profiling studies. After identification and characterization of the respective
deregulated protein spots by mass spectrometry, the quantitative data needs to beconfirmed by a second, independent method like Western blotting.
In a recent proteomics study conducted by the department of radiation
oncology at LMU, Munich, the effect of
γ-irradiation on a human LCL cell line
(lymphoblastoid cell line) was studied by
2-D PAGE. Among many other deregulated proteins, MCM7, a DNA replication factor, was identified to be downregulated by about a factor of two in theirradiated LCL sample.
To validate this result, a quantitative Western blotting experiment wasperformed with monoclonal antibodies
raised against MCM7. Two data-normalization methods were applied andcompared, namely data normalization
with the GAPDH HKP and Stain-Freetechnology.
Figure 2A shows the corresponding high-quality PVDF blot of the LCL

Genetic Engineering and Biotechnology Newssamples before antibody incubation.
Figure 2b illustrates probing the same
PVDF blot for target protein MCM7 andloading control protein GAPDH using amultiplex fluorescent approach. By running a dilution series of the LCL samples
from 10–50 µg per lane, it was verified
beforehand that the 30 µg protein load in
combination with the antibody dilutions
as described in Figure 2B give linear fluorescent signals (data not shown).
Data normalization of the MCM7
signal intensities with GAPDH data andStain-Free data revealed that the downregulation factors of MCM7 are 0.59
with GAPDH (p-value <0.03) and 0.44
with Stain-Free (p-value <0.008). Bothfactors reflect well with the expected
downregulation of about 50% initiallyobtained from 2-D PAGE experiments.
The comparison of raw MCM7 signals(no normalization) reveals a downregulation factor of 0.48 (p-value <0.02) with
35% standard deviation.
Although it is possible to use housekeeping proteins as reference for datanormalization, especially if used in a multiplex fluorescent experiment as described
here, it is highly recommended to consider
Stain-Free technology as a more comprehensive and robust tool for quality management in Western blotting, particularly
when protein deregulation factors aresmall, e.g., in the 10% to 20% range.
ChemiDoc MP Imaging System
Bio-Rad’s latest imaging system, theChemiDoc MP, is designed to enhanceWestern blotting. As Bio-Rad’s mostsensitive imager, the ChemiDoc MP is
designed to meet the imaging needs of amulti-application environment. Capabilities include chemiluminescent and multiplex fluorescent Western blot detection,and gel documentation.
The Stain-Free feature of ChemiDoc
MP enables researchers to take advantage of techniques such as Stain-Free total protein normalization. Whether users
are accustomed to chemiluminescent detection, multiplex fluorescent detection,
or would like to take advantage of Stain-Genetic Engineering and Biotechnology News|Free technology, the versatility of theChemiDoc MP meets the diverse needsof modern laboratories.
Conclusion
The use of Bio-Rad Stain-Free gelsand a Stain-Free enabled imaging system
can vastly improve the Western blot process. Protein separation can be visualizedon the gel after electrophoresis. Proteintransfer to the membrane can be confirmed and assessed prior to blot detection, and blot normalization can occur
without the extra steps required for typical HKP detection. These improvements
enable faster time-to-results and higher
data confidence.
Ryan Short (Ryan_Short@bio-rad.
com) is a marketing manager in the lab
separations division of Bio-Rad Laboratories. Anton Posch, Ph.D. (Anton_Posch@bio-rad.com), is a staff scientist
in the protein technologies R&D groupat Bio-Rad Laboratories.
Web: www.bio-rad.com/ad/mp.

genengnews.com |November 15, 2011