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Dot-it-Spot-it®  Total Protein Assay

We compare the innovative Dot-it-Spot-it Total Protein Assay with two established assays, examining sensitivity, specificity, compatibility, and protein-to-protein variation.

Table of Contents
Sensitivity
Specificity in biological specimens
Specificity and compatibility
Protein-to-protein variation (coming soon)

SENSITIVITY

Sensitivity and sample volume

This study compares the most sensitive variants of three Total Protein Assays to determine their suitability for quantifying protein in minute details of valuable biological samples.

The Dot-it-Spot-it Total Protein Assay was most suitable for this purpose, exhibiting 2000 times greater sensitivity and measuring low protein concentrations using only 0.5 microlitres of the sample.


SPECIFICITY in biological specimens

DNA Cross-Reaction

While total protein assays are effective in measuring protein content, they may also exhibit cross reactions with other biological molecules, such as DNA. These assays can display varying degrees of crossreaction. For instance, in a sample containing 100 μg of DNA, a false-positive result ranging from 15 to 4 μg of BSA could be observed.

SPECIFICITY and COMPATIBILITY

Laemmli buffers

Laemmli Sample Buffer, also known as SDS-PAGE sample buffer, is a widely used reagent for protein sample preparation.
Dot-it-Spot-it Total Protein Assay excels in compatibility with Laemmli buffers, ensuring reliable and efficient protein analysis, see the figure. Notably, the sample can be directly applied without the need for addition of Dilution Buffer (100% sample).
Both Method B and C are unsuitable for detecting low-protein samples in Laemmli buffers as they produce high signals unrelated to protein presence. The false-positive signals have been quantified by comparing them to a BSA standard in TBS, using the unit “BSA-equivalents” (μg/mL).

RIPA and cell lysis buffer

Protein extraction from tissues, cells, or samples typically involves the incorporation of potent detergents and various additives.
Methods B and C display positive signals for both buffers, even in the absence of actual protein. The false-positive signal levels have been compared to a BSA standard in TBS, and the units are expressed as ”BSA-equivalents” (μg/mL). Unfortunately, these methods are not effective for measurements in the low protein range.
The Dot-it-Spot-it Total Protein Assay is designed to prevent false-positive signals. Its adaptability allows it to use both buffers by adjusting the sample percentage in the reaction buffer. For RIPA buffer: Utilize a 4% sample in the reaction buffer and 2.5 μg BSA/mL as the low standard, see the figure. For Cell lysis buffer: Employ a 5% sample in the reaction buffer and 2 μg BSA/mL as the low standard