Radiance chemiluminescent substrate

Manufactured by Azure Biosystems

Radiance Chemiluminescent Substrate is a laboratory reagent used to detect and quantify protein levels in Western blot analysis. It generates a chemiluminescent signal proportional to the amount of target protein present in the sample.

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Lab products found in correlation

4 protocols using radiance chemiluminescent substrate

Mitochondrial Transcription Factor Analysis

Cited 3 times

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Using 4–12% gradient gels, immunoblotting was performed through MES SDS-PAGE, as previously described [21 (link), 26 (link), 29 (link)–31 (link)]. Protein was normalized using the Bradford method. Primary antibodies used in the study included: anti-TFAM, transcription factor A, mitochondrial, 1:500 (SCBT, Dallas, TX), anti-GAPDH 1:1000 (Abcam, Cambridge, MA). The secondary antibody used in the study was a goat anti-mouse IgG (H&L) horseradish peroxidase (HRP) conjugate 1:10,000 (Thermo Fisher). GAPDH expression was used to normalize protein content. Chemiluminescence was measured through Radiance Chemiluminescent Substrate (Azure Biosystems, Dublin, CA), per manufacturer’s instructions and imaged using the G:Box Bioimaging system (Syngene, Frederick, MD). Images were captured through GeneSnap/GeneTools software (Syngene). Densitometry was analyzed using ImageJ and Fiji Software (NIH, Bethesda, MD). Data is represented as optical density with arbitrary units.

Hathaway Q.A., Roth S.M., Pinti M.V., Sprando D.C., Kunovac A., Durr A.J., Cook C.C., Fink G.K., Cheuvront T.B., Grossman J.H., Aljahli G.A., Taylor A.D., Giromini A.P., Allen J.L, & Hollander J.M. (2019). Machine-learning to stratify diabetic patients using novel cardiac biomarkers and integrative genomics. Cardiovascular Diabetology, 18, 78.

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Extracellular Vesicle Protein Profiling

Cited 2 times

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EV pellets were homogenized in RIPA buffer containing 1% SDS, and protein estimated using the BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Western blotting was performed on the various tissue-specific EV as described previously.^{31 (link), 78 (link), 79} Briefly, 20-40 μg protein was loaded onto NuPAGE 4–12% Bis-Tris gels (Invitrogen) and run either under non-reducing (CD63 and CD81) or reducing (Hsp-70 and flotillin-1) conditions followed by their transfer onto nitrocellulose membranes using the iBlot 2 Gel Transfer Device (Invitrogen). Membranes were blocked in TBS SuperBlock buffer (Thermo Fisher Scientific) for 30 min, and immunoblotting carried out overnight at 4 °C with primary antibodies (see Table 1 for details). The next day, membranes were incubated with respective HRP conjugated secondary antibodies for 1.5 hr at room temperature on a rocker. Blots were developed with 1:1 solution of Radiance Chemiluminescent Substrate and Luminol/Enhancer (Azure Biosystems) and visualized using a c300 imaging system (Azure Biosystems). Images were quantified using the ImageJ software.

Chand S., Jo A., Vellichirammal N.N., Gowen A., Guda C., Schaal V., Odegaard K., Lee H., Pendyala G, & Yelamanchili S.V. (2020). Comprehensive Characterization of Nanosized Extracellular Vesicles from Central and Peripheral Organs : Implications for Preclinical and Clinical Applications. ACS applied nano materials, 3(9), 8906-8919.

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Mitochondrial Protein Analysis by Immunoblotting

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Immunoblotting was performed using 4–12% gradient gels through MOPS SDS-PAGE, as previously described [54 (link), 55 (link), 57 (link)–60 (link)]. Normalization of protein content was assessed using the Bradford Method [56 (link)]. Primary antibodies utilized in the study included the following: total OXPHOS Blue Native WV Antibody Cocktail (ab110412) (anti-NDUFA9 (complex I, ab14713, Abcam, Cambridge, MA), anti-SDHA (complex II, ab14715, Abcam), anti-UQCRC2 (complex III, ab14745, Abcam), anti-COX IV (complex IV, ab14744, Abcam), and anti-ATP5A (complex V/ATP Synthase, ab14748, Abcam)), anti-ATP5F1 (complex V/ATP Synthase, ab117991, Abcam), and anti-VDAC (#4866, Cell Signaling Technology, Danvers, MA). Goat anti-mouse IgG (H&L) horseradish peroxidase (HRP) conjugate 1:10,000 (Thermofisher Scientific) and goat anti-rabbit IgG HRP conjugate 1:5000 (Abcam) were used as the secondary antibodies. Normalization of protein content was through VDAC expression. Chemiluminescence quantified with Radiance Chemiluminescent Substrate (Azure Biosystems, Dublin, CA), per manufacturer’s instructions and imaged using the G:Box Bioimaging system (Syngene, Frederick, MD). GeneSnap/GeneTools software (Syngene) was used to acquire images. Densitometry was analyzed using Fiji Software (NIH, Bethesda, MD).

Hathaway Q.A., Majumder N., Goldsmith W.T., Kunovac A., Pinti M.V., Harkema J.R., Castranova V., Hollander J.M, & Hussain S. (2021). Transcriptomics of single dose and repeated carbon black and ozone inhalation co-exposure highlight progressive pulmonary mitochondrial dysfunction. Particle and Fibre Toxicology, 18, 44.

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Quantitative Western Blot Analysis

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Cells were lysed 48 hours post-transfection with 1% SDS and quantified via Pierce Protein Assay (Thermofisher) and a spectrophotometer (Biotek) at 562 nm. Western blots were performed according to the protocols suggested by the producer of each primary antibody and were developed with Radiance chemiluminescent substrate (Azure). Images were taken with an Azure c300 chemiluminescent imaging system and band intensity was quantified using ImageJ. The following primary antibodies were used for western blot analyses: ALKBH5 (Millipore, ABE547), FTO (Millipore, MABE227), and Beta-actin (Sigma, A5441). 25ug of protein was used per lane for all Western blots.

Zepecki J.P., Karambizi D., Fajardo J.E., Snyder K.M., Guetta-Terrier C., Tang O.Y., Chen J.S., Sarkar A., Fiser A., Toms S.A, & Tapinos N. (2021). miRNA-mediated loss of m6A increases nascent translation in glioblastoma. PLoS Genetics, 17(3), e1009086.

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