No-Stain™ Protein Labeling Reagent, 10 reactions - FAQs

iBright Imaging Systems support total lane protein-based normalization and housekeeping gene (HKP)-based normalization. To support the methods best suited for your experiment, iBright Imaging Systems and iBright Analysis Software provide multiple quantitation and normalization options to monitor or mathematically compensate for experimental or sample variability.

To learn more about total lane protein normalization, explore our application note.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The shelf life of the No-Stain Protein Labeling Reagent is at least 2 years from the date of receipt of the product.

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

Even though the No-Stain Protein Labeling Reagent (Cat. No. A44717) is light sensitive, membranes can be kept wet in the dark for at least 3 days with minimal effect on the signal. So, yes, it is possible.

Find additional tips, troubleshooting help, and resources within our Protein Gel Electrophoresis Chambers, Power Supplies, and Accessories Support Center.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.