Protein Analysis by SDS-PAGE and Western Blot

Protein analysis was performed by SDS-PAGE electrophoresis/western blot, loading 30 μg each sample. Proteins were transferred to a nitrocellulose membrane (Amersham, GE Healthcare Europe GmbH, Milano, Italy), and revealed by immunoblotting with specific antibodies: rabbit polyclonal heme oxygenase-1 (HO-1) (1:200)(sc-10789; Santa Cruz Biotechnology™, Dallas, TX, USA), rabbit polyclonal inducible nitric oxide synthase (iNOS) (1:200) (sc-8310; Santa Cruz Biotechnology™), rabbit polyclonal cytochrome 1b1 (Cyp1b1) (1:200) (sc-32882; Santa Cruz Biotechnology™), goat polyclonal heat shock protein 70 (Hsp70) (1:200)(sc-10-70; Santa Cruz Biotechnology™), rabbit polyclonal cyclooxygenase 2 (COX2) (1:1000)(#4842; Cell Signalling Technology^®, Danvers, MA, USA), and rabbit polyclonal myeloperoxidase (MPO) (1:200) (sc-16128-R; Santa Cruz Biotechnology™). The secondary antibodies were appropriate horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5000) (31460 Thermofisher Scientific™ Waltham, MA, USA) or donkey anti-goat (1:2000) (sc-2020; Santa Cruz Biotechnology™). Immunoblot bands have been analyzed and the optical density quantified by LAS4000 (GE Healthcare, Marlborough, MA, USA); all the data have been normalized to Ponceau staining (Sigma Chemical Co., Milano, Italy) [78 (link)].

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Farina F., Lonati E., Milani C., Massimino L., Ballarini E., Donzelli E., Crippa L., Marmiroli P., Botto L., Corsetto P.A., Sancini G., Bulbarelli A, & Palestini P. (2019). In Vivo Comparative Study on Acute and Sub-acute Biological Effects Induced by Ultrafine Particles of Different Anthropogenic Sources in BALB/c Mice. International Journal of Molecular Sciences, 20(11), 2805.

Publication 2019

sc-10789 sc-8310 sc-32882 sc-16128-R sc-2020 LAS4000

Antibodies Cyclooxygenase 2 Cytochrome Donkey Electrophoresis Goat Heat shock protein 70 Heme oxygenase 1 Horseradish peroxidase Immunoblot Inducible nitric oxide synthase Membrane Myeloperoxidase Nitrocellulose Optical Proteins Rabbit Sc 10 Sds page Western blot

Corresponding Organization : University of Milano-Bicocca

Other organizations : San Raffaele University of Rome, University of Milan

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Variable analysis

independent variables

Protein analysis method (SDS-PAGE electrophoresis/western blot)

dependent variables

Protein expression levels of heme oxygenase-1 (HO-1)
Protein expression levels of inducible nitric oxide synthase (iNOS)
Protein expression levels of cytochrome 1b1 (Cyp1b1)
Protein expression levels of heat shock protein 70 (Hsp70)
Protein expression levels of cyclooxygenase 2 (COX2)
Protein expression levels of myeloperoxidase (MPO)

control variables

Loading 30 μg of each sample
Transferring proteins to a nitrocellulose membrane
Using specific antibodies for immunoblotting
Using appropriate HRP-conjugated secondary antibodies
Normalizing data to Ponceau staining

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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