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Immobilon western hrp chemilumiscent substrates

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Immobilon Western HRP Chemilumiscent substrates are a type of lab equipment used to detect and quantify proteins in Western blot analysis. The substrates react with horseradish peroxidase (HRP) conjugated to secondary antibodies, generating a chemiluminescent signal that can be detected and measured.

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Lab products found in correlation

Pvdf membrane, by Merck Group (3 mentions) Pierce bca assay kit, by Thermo Fisher Scientific (2 mentions) Anti mouse or anti rabbit secondary antibody, by Agilent Technologies (1 mentions) Ab9485, by Abcam (1 mentions)

Close spelling variants of this product

Immobilon Western HRP Substrate Immobilon Western substrate Immobilon HRP substrate Immobilon Western HRP substrate Substrates Immobilon

3 protocols using immobilon western hrp chemilumiscent substrates

1

Western Blot Analysis of CISD2 and OXPHOS

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50 µg of total protein extracts, obtained as previously described (40 (link)), were separated on an acrylamide gel by SDS-PAGE and transferred to a PVDF membrane (Millipore, Saint-Quentin, France). Specific proteins were detected by using rabbit polyclonal anti-CISD2 (1/1000, Pierce#PA5-34545) and anti-GAPDH (1/20000, Abcam #ab9485). A cocktail of anti-human total OXPHOS complex antibodies (Mitosciences, Eugene, USA) was used at 1/1000. Anti-mouse or anti-rabbit secondary antibody (Dako) was used at 1/5000 and signals were detected using a chemiluminescence system (Immobilon Western HRP Chemilumiscent substrates, Millipore).
Rouzier C., Moore D., Delorme C., Lacas-Gervais S., Ait-El-Mkadem S., Fragaki K., Burté F., Serre V., Bannwarth S., Chaussenot A., Catala M., Yu-Wai-Man P, & Paquis-Flucklinger V. (2017). A novel CISD2 mutation associated with a classical Wolfram syndrome phenotype alters Ca2+ homeostasis and ER-mitochondria interactions. Human Molecular Genetics, 26(9), 1599-1611.
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2

Protein Quantification and Western Blot Analysis

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The concentration of proteins was determined using the Pierce BCA assay
kit (Thermo Fisher Scientific). 5–25 μg of total protein extracts
or 5–15 μg high-purity mitochondria were separated on
acrylamide-SDS gels and transferred to PVDF membranes (Millipore). Specific
proteins were detected by using different antibodies listed in Supplementary Table 2. Signals were
detected using a chemiluminescence system (Immobilon Western HRP Chemilumiscent
substrates, Millipore). ImageJ was used to quantified protein signals.
Genin E.C., Bannwarth S., Lespinasse F., Ortega-Vila B., Fragaki K., Itoh K., Villa E., Lacas-Gervais S., Jokela M., Auranen M., Ylikallio E., Mauri-Crouzet A., Tyynismaa H., Vihola A., Augé G., Cochaud C., Sesaki H., Ricci J.E., Udd B., Vives-Bauza C, & Paquis-Flucklinger V. (2018). Loss of MICOS complex integrity and mitochondrial damage, but not TDP-43 mitochondrial localisation, are likely associated with severity of CHCHD10-related diseases. Neurobiology of disease, 119, 159-171.
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3

Quantifying Protein Abundance with BCA Assay

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The concentration of proteins was determined using the Pierce BCA assay kit (Thermo Fisher Scientific). 2.5-20µg of total protein extracts were separated on acrylamide-SDS gels and transferred to PVDF membranes (Millipore). Specific proteins were detected by using different antibodies listed in supplementary table 1. Signals were detected using a chemiluminescence system (Immobilon Western HRP Chemilumiscent substrates, Millipore).
ImageJ was used to quantify protein signals.
Genin E.C., Bannwarth S., Ropert B., Mauri-Crouzet A., Lespinasse F., Augé G., Fragaki K., Cochaud C., Lacas‐Gervais S., & Paquis‐Flucklinger V. (2021). SLP2/prohibitins aggregates and instability of the PHB complex are key elements in CHCHD10S59L-related disease.
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