Scripps Laboratories

Technical Brief: BCIP/NBT Western Blot Comparison

In a side-by-side comparison, western blot results demonstrate that alkaline phosphatase substrate BCIP/NBT from Scripps Laboratories works faster and identifies more bands than substrate from 3 leading biochemical suppliers. (view as pdf)   

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INTRODUCTION
Swift, accurate detection of all protein bands upon western blotting is essential to obtaining reliable results. Failure to do so could yield erroneous experimental data and lead to hours of wasted research.

In a side-by-side comparison, the Scripps BCIP/NBT One-Component Substrate was faster and identified more bands than similar BCIP/NBT products from three leading biochemical suppliers.

METHODS
Western blots were run on Prostate Specific Antigen (PSA; cat. no. P0725) under non-reducing and heated conditions. After transfer and blocking, the blot was incubated first with anti-PSA monoclonal antibody, clone BP005S (cat. no. MP077) and then with goat anti-mouse IgG:alkaline phosphatase conjugate. Load sizes for each blot were 0.2 µg (lane 1) and 1.5 µg in (lane 2). The blots were incubated with either Scripps BCIP/NBT One-Component Substrate (cat. no. B0522) or a similar substrate from one of three well-known biochemical suppliers. Photos of each blot were taken at 3 minutes and 5 minutes.
 

RESULTS
As can be seen in the images above, banding visualized using the Scripps substrate at 3 minutes is as clear and well developed as that from the other three substrates at 5 minutes. Furthermore, the Scripps banding is considerably stronger than that from Vendors Y & Z at 5 minutes. In fact, the gels from Vendors Y & Z had to be incubated for 15 minutes before their color was as strong as the Scripps gel at 5 minutes. (Results not shown.)

In addition, the Scripps substrate identified PSA bands not visible in the other blots. Looking closely, higher molecular weight PSA variants are clearly visible in the Scripps blots, but are either non-existent or very faint in the others. This occurred even though all reagents used, except BCIP/NBT, were identical.

DISCUSSION
In the western blot experiment presented here, BCIP/NBT substrate from Scripps provided results that were quicker: 3 minutes vs. as long as 15 minutes. Also, results with the Scripps substrate were more accurate, as higher molecular weight PSA variants were clearly visible, while the substrate from three other suppliers either did not show these variants or stained them only faintly.

Providing reliable data in a timely manner is imperative for reporting accurate scientific results. We conclude the BCIP/NBT substrate from Scripps outperformed that from three leading biochemical suppliers.

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