Table of Contents
Materials and Methods
Preprogramming of the Jenway Genova Nano is done with parameters for a number of protein assays, several of which can be adapted for use with the micro volume accessory. There are a number of benefits of performing scaled down reaction volumes including reduction in reagent and sample volumes and use of fewer consumables such as cuvettes.
Figure 1. Read head of the Genova nano showing a sample in the read position
This article discusses the use of Pierce 660nm protein assay kit using the Genovo Nano. The assay is prepared in a microtitre plate using just 10µl of test sample and has a linear range of 50-2000µg/ml. The assay is easy and quick to perform and is compatible with reducing agents and certain detergents.
Materials and Methods
All reagents were equilibrated to room temperature. The pre-diluted bovine serum albumin standards supplied with the kit was used to prepare a standard curve. A zero (0mg/ml protein, phosphate buffered saline (PBS, pH7.2, Sigma P4244)) standard was also included. 10µl of each standard were pipetted into appropriate wells of a microtitre plate. All the standards were tested three times.
Four unknown protein samples were determined, which include:
- Prostatic acid phosphatase (PAP)
- BSA and
- egg white
All samples were diluted in PBS and were tested with no addition and in the presence of 3mM dithiothreitol (DTT) or 1% SDS. 10µl of each sample were pipetted into appropriate wells of a microtitre plate. All the samples were tested in triplicate.
150µl of reagent was added to each standard and sample in the plate. Gentle shaking was done on a microtitre plate shaker and then incubation was done at room temperature for 5 minutes.
The path length was set to 0.5mm and the Genova Nano was set to the Pierce 660nm assay mode. The number of replicates was set to 3 and manual measurement mode chosen to allow measurement of 3 individual replicates. The number of standards was set to 8 in the quantitation table and the protein standard values including the zero were entered into the table. From the quantitation table, a new standard curve was initiated. Water was used to blank the unit.
Figure 2. Pierce 660nm operating menu.
For measurement, 2µl of each standard were pipette to the read head and the lid was lowered. The sample was wiped off both the bottom and top read heads using a lint-free tissue after each measurement.
After the standard curve is created, each of the unknown samples was measured in the same way. Results were saved to an inserted USB memory stick.
The Genova Nano has several curve fit options. The interpolate curve fit function was used to record the unknown sample concentrations. Figure 3 shows the standard curve plotted using Microsoft Excel and shows a linear response over the standard protein concentration range tested.
Figure 3. Pierce 660nm assay standard curve showing linearity over the complete concentration range tested.
Table 1. provides the derived protein concentration results for the unknowns along with the average absorbance values. The virtual dilution of the sample at the 0.5mm path length causes the absorbance values to be low. There can be a large difference in derived concentration by very small changes in absorbance. The standard deviations of the triplicate samples are very low and typically less than 10% of the total.
Table 1. Protein concentrations of the unknown samples derived from the standard curve on the Genova Nano.
||Average Abs 660nm
|PAP + DTT
|PAP + SDS
|γ-Globulin + DTT
|γ-Globulin + SDS
|BSA + DTT
|BSA + SDS
|Egg white + DTT
|Egg white + SDS
The Pierce 660nm assay is affected by some interfering substances that include ionic detergents such as SDS. This is proved by the results obtained in this assay where the SDS significantly reduced the absorbance of the samples. An ionic detergent compatibility reagent is available as an additive for the kit for use with samples containing SDS. DTT does not affect the kit at the tested concentration and this is illustrated in the results obtained which are comparable to samples which had no DTT added.
Significant cost savings in terms of sample, time and consumables are possible by the ability of the Genova Nano to measure small sample volumes pipetted directly onto the read head. This article shows that a standard cuvette-based protein assay can be adapted for micro volume use with minimal modification and produces reproducible results.
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