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Gene & Protein in Disease Immunoblotting large and small proteins

NaCl, 50 mM Tris-HCl, pH 8.0) supplemented with larger pore nitrocellulose membrane. We expect that these
protease and phosphatase inhibitor (Thermo Scientific, steps may help other researchers detect small and large
1861281), followed by denaturation in LDS sample proteins, which may contribute to increasing knowledge of
buffer (ThermoFisher) supplemented with 20 mM DTT molecular biology.
since the traditional homemade loading buffers are not
suitable due to its high amount of SDS. Protein extracts Acknowledgments
are boiled at 72°C for 5 min. Protein standards should also We thank Manel Esteller and Montse Sanchez-Céspedes
be considered since most of the protein standards do not for their guidance of SVIP and MGA western blot
include molecular weights <250 kDa. For this purpose, experiments, respectively. We also thank Paula López-
a protein marker reagent containing molecular weights Serra and Lida Roselló-Tortella for technical assistance on
up to 460 kDa (HiMark Pre-stained Protein Standard, SVIP western blot optimization.
™
ThermoFisher) is selected.
Electrophoresis is performed in 4 – 12% NuPage Funding
Bis-Tris 1.0 mm width gel in MOPS SDS running buffer This work was supported by Spanish Ministry of Economy
(Novex , Life technologies). Protein transfer is the biggest and Competitiveness FPI Fellowship BES-2015-072204 to
™
challenge for high-molecular-weight proteins. For this Paula Llabata and BES-2015-073053 to Pere Llinàs-Arias.
case, the amount of input protein for the western blot
should also be adjusted. Any amount below 30 µg may Conflict of interest
lead to an undetectable band after transfer. Therefore, The authors declare they have no competing interests.
40–50 µg of input protein is loaded per well. Proteins are
then transferred to a nitrocellulose membrane (0.45 µm, Author contributions
™
Amersham Protran NC) in Tris-glycine transfer buffer
™
(Thermo Scientific) supplemented with 10% methanol and Conceptualization: All authors
incubated overnight at 4°C at a constant voltage of 35 V. Methodology: All authors
As mentioned above, the amount of protein needed for Writing – original draft: All authors
the detectable band after transference is higher than the Writing – review & editing: All authors
amount needed in the SWP. Moreover, the exposure time Ethics approval and consent to participate
for obtaining a clear band may also be higher. Therefore, the
development of western blot images is also challenging. For Not applicable.
these reasons, the chemiluminescence method is discarded
to avoid membrane burning. Instead, images are taken Consent for publication
with the Odyssey Imaging Systems, whose developing Not applicable.
®
method is based on fluorescence. The application of these
SWP variations did not affect the determination of smaller Availability of data
proteins, such as MYC-associated protein X (MAX, 25 kDa), Raw western blots images can be found in supplementary file.
which was detected on the same nitrocellulose membrane.
This procedure efficiently detected Brahma-related gene 1 References
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The above-mentioned modifications overcome the https://doi.org/10.1038/nature11247
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detection requires NP40 lysis buffer, electrophoresis using 4. Llinàs-Arias P, Rosselló-Tortella M, López-Serra P, et al.,
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Volume 2 Issue 2 (2023) 4 https://doi.org/10.36922/gpd.0547