AdvanStain Total Fluorescent Protein Staining Kits

AdvanStain Total is a fluorescent protein stain for PVDF and Nitrocellulose membranes that allows for sensitive and quantitative visualization of protein bands.

For precise quantitative Western blot data, it is critical to perform normalization to account for sample inconsistencies, sample loading errors and uneven transfer. In an effort to increase transparency, total protein normalization is quickly becoming the standard for Western blot normalization as it is now the preferred method for top tier journals. The AdvanStain Total Fluorescent Protein Staining Kits provide a convenient method for total protein normalization that is compatible with digital imaging systems equipped with a Cy3‑compatible filter.

AdvanStain Total Fluorescent Protein Staining Workflow

AdvanStain Total spectrum

Figure 1. AdvanStain Total can be imaged with a CCD or laser based imaging system using green-light excitation.

Broad linear range

Figure 2. AdvanStain Total has a broad linear range. Untreated Hela lysate was serially diluted 1:2 prior to separation by SDS-PAGE. After electrophoresis the proteins were transferred to a PVDF membrane for total protein staining (a). The image was quantified, and staining intensities analyzed by linear regression (b).

Fluorescent Western blotting

Figure 3. AdvanStain Total is compatible with IR fluorescent Western blotting, without destaining. Untreated and IFNα treated HeLa lysate was loaded in quadruplicate then transferred to a PVDF membrane using a wet transfer. After transfer, the membrane was stained with AdvanStain Total and imaged using a Cy3 filter (a). After staining, the blot was blocked and probed for GAPDH and phosphorylated STAT3. SpectraDye-IR700 and IR800 secondary antibodies were used for detection (b).

Chemiluminescent Western blotting

Figure 4. AdvanStain Total is compatible with chemiluminescent Western blotting, without de-staining. Untreated and IFNα treated HeLa lysate was loaded in quadruplicate then transferred to a PVDF membrane using a wet transfer. After transfer, the membrane was stained and imaged using a Cy3 filter (a). After staining the blot was blocked and probed for Actin and phosphorylated STAT3. The antibodies were detected with a goat anti-rabbit HRP conjugate and WesternBright-ECL HRP Substrate (b).

Maximum flexibility

Figure 5. AdvanStain Total may be imaged wet or dry without increasing general background. Dilutions of untreated HeLa lysate were electrophoresed on an SDS-PAGE gel then transferred to a PVDF membrane using a wet transfer. After transfer, the membrane was partitioned and stained. AdvanStain Total was imaged in the Cy3 channel wet and dry (a), the competitor stain was imaged in the Cy5 channel wet and dry (b).

 Click here to request a sample for PVDF membrane

 Click here to request a sample for NC membrane

• AdvanStain Total brochure
• AdvanStain Total-PVDF short protocol
• AdvanStain Total-NC short protocol
• AdvanStain Total Staining Solution SDS
• AdvanStain Total Washing Solution SDS
• Total Protein Stain Selection Guide
• Advansta’s Step-by-Step Guide to Western Blots