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AZURE Q MODULE

Optional Green Fluorescence Channel

Add Total Protein Normalization to ECL or NIR Western Blots

Why Add a Q Module?

Publication guidelines are becoming increasingly strict: accurate quantitation of Western Blots requires normalization to an internal loading control. One issue is that housekeeping proteins have poor linearity at high abundances. OUR SOLUTION? A custom workflow for Total Protein Staining (TPS).

The Azure Q Module is an optional fluorescence channel intended for detecting TPS such as TotalStain Q. With an extra channel, you can still detect up to two proteins of interest + total protein.

The wider linearity and dynamic range of TPS is the new standard preferred by journals for accurate Western Blot quantification.

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Azure 300Q

Enjoy the sensitivity of chemiluminescence with the linearity and quantitative potential of fluorescent total protein stains such as Azure TotalStain Q.

Normalize signal in your chemiluminescent western blots with the addition of a fluorescent channel. Detect more than just housekeeping proteins for quantitative western blots on top of your traditional ECL detection.

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Azure 500Q

Break through the preconceived limits of two fluorescent channels with the Azure Q module.

Near-infrared lasers provide the highest signal to noise ratio
with minimal crosstalk. The addition of a third fluorescent
channel to the camera-based imager allows the simultaneous
detection of housekeeping proteins or a total protein stain

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Sapphire NIR-Q

Why limit your scanning capabilities to two channels?

With the Sapphire NIR-Q scanner, target two proteins of
interest in the 700 and 800nm channels and add a third target
protein OR total protein stain using the Q module (520nm).

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TotalStain Q

TotalStain Q solves the issue of housekeeping proteins that lack linearity and normalization accuracy at high protein loads.

TotalStain Q is a total protein staining reagent, compatible with chemiluminescent or near infrared fluorescent Western Blot detection. The linear range between 1–50 μg of lysate makes it ideal for normalization of low expressed proteins that require high loads of protein to reach the sensitivity for the detection of the target.

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Imager Models

200 | 280 | 300 | 400 | 500 | 600 | Sapphire

Request pricing, product specs, arrange a demo, or more information:

    Significance of Western Blot Normalization​

    Quantitative western blots require the normalization of signal to an internal loading control. Guidelines from leading journals state that total protein normalization is preferred over normalization to housekeeping proteins.

    Journal of Biological Chemistry
    Transparency is the Key to Quality

    “It is not uncommon to “correct” Western blot signals for protein loading by normalizing to [housekeeping proteins present in the same replicate], e.g.: β-actin, α-tubulin, transferrin, GAPDH, HPRT1. The problem with this approach is that a linear relationship between signal intensity and the mass or volume of sample loaded must be confirmed for every antigen… It is typically better to normalize Western blots using total protein loading as the denominator [given its wider linear range].”.

    Fosang, Amanda J, and Roger J Colbran. “Transparency Is the Key to Quality.” The Journal of biological chemistry vol. 290,50 (2015): 29692-4. doi:10.1074/jbc.E115.000002

    Read Full Article

    Journal of Proteomics
    Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

    “Total protein staining (TPS) represents the actual loading amount more accurately than HKPs due to minor technical and biological variation. Further, the broad dynamic range of TPS solves the issue of HKPs that commonly fail to show loading differences above small loading amounts of 0.5-10 μg.”

    Moritz CP. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics. 2017 Oct;17(20). doi: 10.1002/pmic.201600189. PMID: 28941183

    Read Full Article

    Documents

    Document TypeDescription
    Application NoteThree-Color Western Blots with AzureSpectra AntibodiesDOWNLOAD
    Application NoteImaging Three-color Western Blots with the Azure c600DOWNLOAD
    Application NoteAccurate Western Blot Normalization with AzureRed Fluorescent Protein StainDOWNLOAD
    Application NotePhosphorylated Protein Detection is More Efficient by Fluorescent Western BlotDOWNLOAD
    Application NoteIncreasing Assay Efficiency with Four Color DetectionDOWNLOAD
    BrochureThe Azure Q ModuleDOWNLOAD
    BrochureSapphire Applications OverviewDOWNLOAD
    BrochureSapphire Biomolecular ImagerDOWNLOAD

    citations

    93/100
    Bioz Stars Product Rating
    The world's only objective ratings for scientific research products
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    99 Product Citations

    The molecular tweezer CLR01 improves behavioral deficits and reduces tau pathology in P301S-tau transgenic mice.

    Alzheimers Res Ther 2021 4 Jan
    ‘washed thrice in TBS-T and incubated with HRPconjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology) diluted 1:10,000 in blocking solution for 1 h at room temperature. … Dots were visualized by incubating the membranes with ECL substrate (SuperSignal, West Pico PLUS, ThermoFisher) for 5 min and imaged using an ([read more]) Azure 300 Chemiluminescent Western Blot Imaging System ( Azure biosystems ).. Native PAGE and western blots Soluble fractions were thawed on ice and mixed with 4× NativePAGE sample buffer comprising 50 mM Bis-Tris, 50 mM NaCl, 10%.. ‘ [/read]

    Targeting Scavenger Receptor Type B-1 (SR-B1) and Cholesterol Inhibits Entry of SARS-CoV-2 Pseudovirus in Cell Culture

    bioRxiv 2020 14 Dec
    PrePrint: washed 10 minutes (3X) in TBST (0.1% Tween-20) secondary goat anti-rabbit antibody (BioRad, 1721019) was applied (1:1000) for 1 hour at R.T. and blot was washed (3X) same as described above. … Protein was detected using enhanced chemiluminescence (ECL) detection (Bio-Rad, 1705060) and an Azure 300 ( Azure Biosystems ) gel imaging system.’

    Hyperoxia induces endoplasmic reticulum stress-associated apoptosis via the IRE1α pathway in rats with bronchopulmonary dysplasia

    Mol Med Rep 2020 9 Nov
    ‘Following the primary incubation, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; goat anti-mouse and anti-rabbit IgG-HRP; cat. nos.. SA00001-1 and SA00001-2, respectively; Wuhan Sanying Biotechnology) at room temperature for 2 h. Proteins bands were detected using the Super ECL Plus (Thermo Fisher Scientific, Inc.) ([read more]) and luminescence analysis was performed with a chemiluminescence imaging system ( C300 ; Azure Biosystems, Inc .).. The bands were standardized according to β-actin. Chemiluminescence Module , is1003 , is1003 , chemiluminescence module D049449 , Chemiluminescence imaging.. ‘ [/read]

    Autoimmunity linked protein phosphatase PTPN22 as a target for cancer immunotherapy

    J Immunother Cancer 2020 30 Oct
    ‘Membranes were blocked with 5% milk and 0.2% Tween in PBS.. HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, Massachusetts, USA), ECL reagent (Bio-Rad, Hercules, California, USA) and Azure 600 (Azure Biosystems , Dublin, California, USA) for band visualization were used in Western blot analysis.’

    Chronic Alcohol Exposure Induces Aberrant Mitochondrial Morphology and Inhibits Respiratory Capacity in the Medial Prefrontal Cortex of Mice

    Front Neurosci 2020 22 Oct
    ‘were incubated (5% BSA) for 1 h at room temperature with respective anti-rabbit and anti-mouse secondary antibodies (1:1000, Millipore). … Blots were visualized with the Radiance Q Chemiluminescent Substrate (Azure Biosystems, Dublin, CA, United States), ([read more]) developed on an Azure 300 Chemiluminescent Western Blot Imaging System ( Azure Biosystems , Dublin, CA, United States), and band optical density quantification was performed using NIH ImageJ software.’ [/read]

    Involvement of Mitochondrial Mechanisms in the Cytostatic Effect of Desethylamiodarone in B16F10 Melanoma Cells

    Int J Mol Sci 2020 5 Oct
    ‘The films were scanned, and the pixel densities of the bands were determined using the NIH ImageJ software.. Alternatively, chemiluminescence was measured on an Azure 300 ( Azure Biosystems ) imaging system that digitized the bands’ chemiluminescence intensities using its inbuilt software.. For stripping and reprobing, the membranes were washed in stripping buffer (0.1 M glycine, 5 M MgCl2, pH 2.8) for 1 h at room temperature.’

    The influence of PI3K inhibition on the radiotherapy response of head and neck cancer cells

    Scientific Reports 2020 1 Oct
    ‘(#9271), mTOR (#2972), phospho-mTOR ser2448 (#2971), p70S6 (#9202), phospho-p70S6 (#9205), 4EBP1 (#9452) and phospho-4EBP1 (#9459) from Cell signalling technology, Vinculin (Sigma-Aldrich) was used as loading control. … After incubation with a secondary antibody, ([read more]) visualization was performed with enhanced chemiluminescence reagent (ECL Ultra, Perkin Elmer) using a chemiluminescence imager ( c600 Azure Biosystems , CA, USA).. Densitometry analysis was performed using ImageJ2 software.’ [/read]

    4-HNE Immunohistochemistry and Image Analysis for Detection of Lipid Peroxidation in Human Liver Samples Using Vitamin E Treatment in NAFLD as a Proof of Concept.

    J Histochem Cytochem 2020 1 Sep
    ‘Membrane was blocked for 1 hr in 5% milk in Tris-buffered saline Tween-20 (TBST) and HNEJ-2 antibody was incubated overnight (1:1000) at 4C with gentle agitation.. Bands were visualized using enhanced chemiluminescence (ECL; Thermo Fisher Scientific), ([read more]) and band intensity was recorded with an Azure 300 ( Azure biosystems ).. Band volume intensities were quantified with Fiji20 and relative concentrations of 4-HNE protein adducts were calculated as the ratio of adducts to the amount of β-actin (Cell Signaling Technology).'[/read]

    Plant NLR targets P-type ATPase for executing plasma membrane depolarization leading to calcium influx and cell death

    bioRxiv 2020 31 Aug
    PrePrint: Resuspended cell suspension were incubated for 1∼3 hr at room temperature and pressure-infiltrated with a 1 mL needless syringe into N. benthamiana leaves.. To visualize cell death, the leaves were photograph after 3∼5 days after infiltration using Cy5 and Cy3 channels in Azure 400 ( Azure biosystem ).. Constructs Clones for epitope tagged proteins were generated using ligation-independent cloning method .’

    Cdkn2a loss in a model of neurofibroma demonstrates stepwise tumor progression to atypical neurofibroma and MPNST.

    Cancer Res 2020 19 Aug
    Abstract: Plexiform neurofibromas (PN) are benign nerve sheath Schwann cell tumors characterized by biallelic mutations in the NF1 tumor suppressor gene.’

    Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling

    Cell Rep 2020 11 Aug
    ‘4%–15% precast polyacrylamide gel (Bio-Rad,456–1084), transferred to 0.45 μm PVDF membrane, blocked with 5% milk (Bio-Rad,170–6404) for 1h, and incubated with primary antibody ( ) at 4°C overnight. … After incubation with HRP-conjugated secondary antibody for 1 h at room temperature, signals were detected using Azure 300 ([read more]) (Azure biosystems ) and images were taken with cSeries Capture Software.. RNA sequencing Incisor samples taken one week after induction of one-month-old Gli1 -Cre ERT2 ;Runx2 fl /fl and Runx2 fl /fl mice were dissected.'[/read]

    Robust Survival-Based RNA Interference of Gene Families Using in Tandem Silencing of Adenine Phosphoribosyltransferase 1Robust Survival-Based RNA Interference of Gene Families Using in Tandem Silencing of Adenine Phosphoribosyltransferase 1 [OPEN]

    Plant Physiol 2020 6 Aug
    ‘0.1% (v/v) Tween 20, incubated in secondary antibody (goat anti-rabbit for XIa-CCT, goat anti-mouse for α-tubulin) at 1:100,000 dilution for 1 hr at room temperature, then washed a final three times. … Blots were developed using homemade enhanced chemiluminescence reagent, and chemiluminescent images were acquired using an Azure 600 ( Azure Biosystems ).. Densitometry was performed using ImageJ ( ) to allow comparison of relative protein abundance.’

    Cytoprotective effects of fermented oyster extracts against oxidative stress-induced DNA damage and apoptosis through activation of the Nrf2/HO-1 signaling pathway in MC3T3-E1 osteoblasts

    EXCLI Journal 2020 4 Aug
    ‘After washing with PBS, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at RT.. Protein bands were detected using an ECL detection system and chemiluminescence imaging (Azure Biosystems, Inc., Dublin, CA, USA).. ([read more]) Small interfering RNA (siRNA) transient transfection siRNA transient transfection for Nrf2 gene inhibition was performed using Lipofectamine 2000 reagent, according to the manufacturer’s instructions.’ [/read]

    Mixed plasticizers aggravated apoptosis by NOD2-RIP2-NF-κB pathway in grass carp hepatocytes.

    J Hazard Mater 2020 21 Jul
    Abstract: The wide application of plastics led to the wide exposure of plasticizers to the environment.’

    Engineering Light-Responsive Contractile Actomyosin Networks with DNA Nanotechnology.

    Adv Biosyst 2020 21 Jul
    ‘The gel was run in a Xcell SureLock Mini Cell (Thermo Fisher) for 35 min at 200 V in a 1× NuPAGE SDS MES running buffer and afterward stained with InstantBlue (Expedeon).. The gel was imaged with either an Amersham Imager 600 (GE Healthcare Life Sciences) or Azure 600 (Azure Biosystems ).. ([read more]) Design and Assembly of the DNA Linkers: The design and assembly of the DNA linkers was adapted from Jahnke et al.[35] In brief, a set of random fixed-length DNA sequences was generated in.. ‘ [/read]

    In vitro Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization

    Front Cell Dev Biol 2020 16 Jun
    ‘The membranes were once again washed and prior to their revealing, 500 μL of a 1:1 mixture containing Detection buffer C and D was added for 2 min at RT.. Finally, the chemiluminescence image of each membrane was obtained using a Sapphire Biomolecular Imager ([read more]) ( Azure Biosystems , United States).. The intensity of each dot was quantified using the AzureSpot software (Azure Biosystems, United States) by designing an 8 × 8 dot grid adjusted to include each individual point.’ [/read]

    Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16

    Anal Bioanal Chem 2020 3 Jun
    ‘The DNA was purified using phenol-chloroform extraction and ethanol precipitation.. Gel imagingTo identify 5′-FAM-specific LAMP products, unstained agarose and acrylamide gels were imaged under 488-nm laser excitation using the Sapphire Biomolecular Imager ( Azure Biosystems , Dublin, CA).. Images were processed with Sapphire Capture software (Azure Biosystems, Dublin, CA).’

    Robust survival-based RNAi using in tandem silencing of adenine phosphoribosyltransferase

    bioRxiv 2020 10 Apr
    PrePrint: were washed three times in TBS-T, incubated in secondary antibody (goat anti-rabbit for XIa-CCT, goat anti-mouse for α-tubulin) at 1:100,000 dilution for 1hr at room temperature, then washed a final three times. … [read more])Blots were developed using homemade ECL reagent and chemiluminescent images were acquired using an Azure 600 ( Azure Biosystems ).. Densitometry was performed using ImageJ ( ) to allow comparison of relative protein abundance.’ [/read]

    The role of activated NLRP3 inflammatory body in acute kidney injury in rats caused by sepsis and NLRP3-TXNIP signaling pathway

    Saudi J Biol Sci 2020 18 Mar
    ‘PBS solution and protein labeling reagent were used to label the tissue.. The optical densities of NRLP3, ASC, Caspase-1, and IL-1β were analyzed with Chemiluminescence Imaging and Analysis System ( Azure Biosystems Inc .,([read more]) Dublin, CA, USA).. 2.5 Detection of NLRP3/ASC/Caspase-1 p45/Caspase-1 P10 expression by immunoblotting The obtained slices were placed in radioimmunoprecipitation assay (RIPA) buffer, then treated at low temperature, and further processed by low temperature.. ‘ [/read]

    SWI/SNF chromatin remodeling complex and glucose metabolism are deregulated in advanced bladder cancer, et al. SWI/SNF chromatin remodeling complex and glucose metabolism are deregulated in advanced bladder cancer

    IUBMB Life 2020 19 Feb
    ‘After incubation with primary antibodies, membranes were washed three times for 5 min in TBS‐T and probed with secondary antibodies (Supplementary Table ) for 1 hr at RT.. Chemiluminescence signal was detected by WesternBright Quantum detection kit (Advansta) using Azure Biosystems ([read more]) chemiluminescence detection system .. 2.7 Transcriptome database analysisThe microarray datasets obtained from Gene Omnibus Database (GEO) with accession number GSE31684 from 93 BC samples , were reanalyzed using GeneSpring GX software (Agilent) according to the.. ‘[/read]
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