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User Guide: No-Stain Protein Labeling Reagent Manual / Product Insert

  • Version: B.0
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Can proteins in a gel be transferred to a membrane after the gel has been labeled using the No-Stain Protein Labeling Reagent? Product FAQ

Answer

We do not recommend labeling a gel with No-Stain Protein Labeling Reagent and then using that same gel for transfer. However, proteins on a membrane transferred from an unlabeled gel can be visualized using the No-Stain Protein Labeling Reagent.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17974

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How does the No-Stain Protein Labeling Reagent work? Product FAQ

Answer

The No-Stain Protein Labeling Reagent provides two active ingredients that result in a fluorophore being covalently attached to the side chains of some of the lysine residues of a protein. One of these active ingredients is contained within the No-Stain Derivatizer and consists of a fluorogenic molecule that does not fluoresce unless it undergoes a specialized reaction with the active ingredient contained within the No-Stain Activator.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17979

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Can I get uniform labeling of proteins when using the No-Stain Protein Labeling Reagent? Product FAQ

Answer

Yes, the No-Stain Protein Labeling Reagent provides uniform labeling of proteins in gels or on membranes, provided they are imaged the same way.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17983

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Can I use the No-Stain Protein Labeling Reagent with any gel type? Product FAQ

Answer

Yes, the No-Stain Protein Labeling Reagent can label proteins in any gel type (i.e., precast gels or 'pour your own' gels) and any gel chemistry (i.e., Bis-Tris, Tris-Glycine, Tris-Acetate, and Tricine gels).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17975

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Can I use any imager to visualize the signal from proteins labeled using the No-Stain Protein Labeling Reagent? Product FAQ

Answer

The No-Stain Protein Labeling Reagent is compatible with a wide-range of imagers that have a UV, Green LED, or fluorescence light source, for example, the iBright imagers.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17976

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Can I use the No-Stain Protein Labeling Reagent with an orange or red dye (i.e., will the signal appear in channel 2, 3, 4 or 5, not just channel 1 in the iBright imager)? Product FAQ

Answer

Channels 3, 4 and 5 on the iBright imager can be used for multiplexing without interference while using the No-Stain Protein Labeling Reagent. Channels 1 and 2 on the iBright imager will show interference. Fluorophores for multiplexing that are compatible with the No-Stain Protein Labeling Reagent are listed here, under the “Secondary Abs” tab (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/total-protein-normalization.html).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17978

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Can I get a similar signal intensity from two same-type gels or two same-type membranes that I label using the No-Stain Protein Labeling Reagent? Product FAQ

Answer

Yes, reproducible signal intensity will be obtained when using the No-Stain Protein Labeling Reagent to label either the same type, identically loaded gels or the same type membranes from the transfer of identically loaded, same-type gels: quantitation results will be similar between the gel pairs and the membrane pairs.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17982

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Will the presence of molecules that are covalently added on the protein through using the No-Stain Protein Labeling Reagent affect the ability of the labeled protein to bind antibody and thus impact the resultant immunodetection signal? Product FAQ

Answer

During the development of the No-Stain Protein Labeling Reagent, over 100 different antigen-antibody pairs were evaluated for impact of using the reagent. The binding of some antibodies to labeled antigens appeared to be impacted compared to the binding to corresponding unlabeled antigens. However, a complete loss of immunodetection signal was not observed for any of the labeled antigens tested.

For the affected antibodies, the immunodetection signal was either higher or lower compared to unlabeled antigens. For most cases in which a lower immunodetection signal was observed due to labeling, a slight increase in exposure time during imaging compensated for the reduced signal; when using smart or automatic exposure settings on an imager, this increase in exposure time was self-adjusting, showing that the labeling did not cause a significant impact on the result.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17972

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Can I perform data analysis with the No-Stain Protein Labeling Reagent if I don't have an iBright imager? Product FAQ

Answer

You should be able to use the same software that you are currently using for quantitative westerns. If you are measuring the signal intensity of a target protein or a housekeeping protein with software such as ImageJ or OpenLab, then you can use that same software for measuring the intensity of No-Stain Labeling Reagent-labeled protein bands.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17980

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What are the excitation and emission maxima of the fluorophore used in the No-Stain Protein Labeling Reagent? Product FAQ

Answer

The fluorophore’s excitation and emission maxima are ~488 nm and 590 nm, respectively. The fluorophore’s spectra can be accessed here, under the “Secondary Abs” tab (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/total-protein-normalization.html).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17977

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Can I use the No-Stain Protein Labeling Reagent to label a gel for visualizing total protein? Product FAQ

Answer

Yes, the protocol in the User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018742_NoStain_LabelingReagent_UG.pdf) provides instructions on how to do this.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17973

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Application note: Easy and accurate normalization of western blot data using No-Stain Protein Labeling Reagent and iBright 1500 Series Imaging Systems Product Literature

Are you planning to develop additional data analysis software for the iBright imager? Product FAQ

Answer

The iBright imager now has software enabling Total Protein Normalization when using the No-Stain Protein Labeling Reagent. Additional data analysis software for the iBright imager is not being planned at this time.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E17981

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Application Note: Easy and accurate normalization of western blot data using No-Stain Protein Labeling Reagent and iBright 1500 Series Imaging Systems (Chinese) Product Literature

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