Colorimetric investigations of soil extracts are also strongly affected by physical interferences scattering , and physico-chemical effects from suspended clays sorption. In the aforementioned study, the Lowry assay was the only method to give correct protein estimates. This introduces limitations regarding the number of samples measurable per run, reducing throughput and speed. The citrate extraction technique described by Wright and Upadhyaya is widely practiced, with protein content commonly referred to as glomalin related soil protein GRSP.
Furthermore, the interference from polyphenols is not quantified in the assay procedure. This means that no distinction can be made between the protein and polyphenolic content, and thus references to GRSP or even protein quantified using the Bradford assay can be very misleading Nannipieri and Eldor, The sensitivity of the Lowry assay to citrate might first appear prohibitive.
Although this has been successful for biofilm extracts of waste-water sludges, it has not previously been tested with soil extracts.
Increases in total protein content were measured by addition of known quantities of BSA to citrate extracts of 3 contrasting soils. Our aims were to:. Soil 1 is classified as a flinty clay loam over clay with sandy inclusions Batcombe series. Soils were sampled both from a bare fallow management area Soil 2 and from permanent grassland Soil 3. Composite soil samples were collected in April using a 2.
A model polyphenol humic acid; Sigma H and protein bovine serum albumin; Sigma A were used throughout as standards. Soil extracts 1, 2 and 3 were diluted 7. The principle of this modification is that the omission of copper sulphate from the reagent enables determination of the auto-absorbance from humic compounds and chromogenic amino acids.
Lowry reagents were made from three stock solutions at 3. The three solutions were combined sequentially in proportions of 1: The second reagent Reagent B was made in the same way, except the copper sulphate solution was excluded and volume substituted with deionised water.
Reagent B was added to plate B in the same way. Two absorbencies per sample were thus obtained: The citrate concentrations investigated were therefore 1, 2, 4 and 5 times the typical assay concentration. The modified Lowry procedure described in paragraph 2. The effect of increasing additions of citrate upon Abs protein over all additions of BSA and citrate is presented for the different analytical matrices.
The known additions of BSA were compared with the measured increase in protein concentration of the samples. Regression analysis was used to compare the strength of the impact of HA additions on the intercept and slope of the calibration in buffer and soil extracts.
The resulting calibration curves of Abs protein against BSA additions to soil extract and buffer plus HA were thus almost indistinguishable at the highest and lowest concentrations of citrate tested Fig. Regression coefficients of multiple regressions for citrate additions given with standard error s. The extent is negligible however in the range of citrate concentrations likely to be used for soil extraction Fig.
Similarly, in soil extract, Abs protein is 0. The suppression of colour development due to protein Abs protein was more fully investigated in the following experiment by varying concentration of HA, both in PBS and soil extracts.
Regression coefficients of Abs protein as f BSA in the three contrasting matrices, also in the presence of citrate. The modified two-to-three-reagent Lowry system was less time-sensitive than the Bradford thus permitting full use of the 96 well plates. The Lowry-based analytical procedure including mixing of stock reagents was achieved in approximately one hour. Although increasing inclusions of HA caused an additive effect in terms of total absorbance, an increasingly large underestimate of the additional protein also occurred, as seen by the reduction of slope angle with increasing HA content.
The Lowry microplate method gave more accurate estimates of the quantity of protein added Fig. With the Lowry microplate assay, addition of HA alone did not result in a false positive indication of protein Fig. Using the Bradford method, the mean squared errors of prediction MSEP of protein added to buffer affected by additions of HA were calculated using the method of Wallach and Goffinet Using the Lowry-microplate technique, the observed values protein measured differ much less from the expected protein added , with corresponding MSEP's of 2, 81, , and , respectively.
The protein concentrations in diluted extracts of soil 1 given by the Bradford and Lowry microplate methods Fig. Using the Bradford assay Fig. In contrast, with the modified Lowry assay Fig. Using extracts of Soil 1, the increases in protein estimate in response to known additions of BSA are presented in Fig. Colour development due to pre-existing soil protein or directly from the added polyphenol is excluded. This would be especially problematic if quantifying extract protein using a calibration curve generated in PBS common procedure.
With the Bradford assay, linear regressions of the responses to added protein, by comparison to the 1: Polyphenol HA suppresses colour development from protein in a Bradford and b Lowry analyses of soil 1 extract. In contrast, using the Lowry microplate assay Fig. Whereas linear regressions of absorbance increases using the Bradford assay accounted for S2a , with Similarly, linear regressions of absorbance increases with the Lowry microplate assay accounted for However, although Lowry responses to HA additions to buffer are, strictly speaking, best described by a 2nd order polynomial Fig.
The Lowry microplate assay thus gave a more linear response than the Bradford assay to both polyphenol and protein additions to soil extracts. It is important to remember that although colour development from protein per se using the Bradford assay is more suppressed by the presence of polyphenol, it is variably compensated for by the colour development directly from the polyphenol itself. This most likely explains the apparent overestimation of protein in soil 3.
In contrast, there appears to be negative bias in soils 1 and 2 by comparison to concentrations given by the Lowry assay.
This is most likely due to suppression of colour development from protein as demonstrated in Fig. Extract protein concentrations corrected for dilution. Modified Lowry also provides estimate of phenolics as HA equivalent: This most likely explains the apparent overestimation of protein in soil 3 by the Bradford assay in comparison to the Lowry.
Overestimation was seen previously with soil extracts containing a large phenolic fraction e. The phenolic content of soil is of further contemporary interest as it has been linked to soil organic matter dynamics in the context of land-use change, climate, and CO 2 emissions, e. Citrate extracts are best thought to contain a mixture of biochemicals from soil microbes, humified soil organic matter, and reaction products of extraction Nannipieri and Eldor, In the current study, depending on soil type, estimates of polyphenol content shown as HAE were between 5 and 10 times greater than the protein content.
It is not possible to comment precisely upon the accuracy of the determinations of soil extract protein per se soil extract in the absence of any BSA or polyphenol additions , because there is currently no universally accepted method to measure protein in soils, with each method being subject to idiosyncratic artefacts Nannipieri and Eldor, Therefore, in this study, comparison of accuracy is based upon the assumption that BSA, the most commonly used protein reference standard, is a good model for soil protein.
It follows that if the Bradford reactive fraction is to serve as a surrogate measure of aggregate stability, or organic matter content, then the de facto GRSP measure may still be useful. Moreover, confounding these pools through use of the Bradford assay will cloud interpretations of the respective contributions of these organic fractions to soil properties.
It is likely that in future, a rapid measure of soil protein distinct from highly humified pools will be called for, e. The structural roles of extracellular proteins are currently being explored in related scientific disciplines and are thought to help impart strength and elasticity to biofilms Flemming and Wingender, A firm causal link between AMF and aggregate stability was later established through other methods, e.
A greater understanding of community-wide extracellular proteinaceous material in soils is now required, and besides improved specificity of extraction methods, the single most important step is likely to be avoiding the largest known artefact currently affecting colorimetric analyses, i.
The modified Lowry assay presented here provided a reasonable estimate of polyphenolic content and a more accurate estimate of protein content in citrate extracts of 3 contrasting soils, and model extracts. It is therefore of potential value in comparative studies of total extractable protein where the polyphenol content is expected to be high. The following are the supplementary data related to this article:.
Effect of citrate upon Lowry estimation of a standards added to PBS, b standards added to soil extract 1. National Center for Biotechnology Information , U. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content.
The method is sensitive down to about 0. Prepare immediately before use by mixing the following three stock solutions A, B, and C in the proportion Folin reagent commercially available. Use a stock solution of standard protein e.
Prepare standards by diluting the stock solution with distilled water as follows: Let the solution stand at room temperature for 10 min. Plot a standard curve of absorbance as a function of initial protein concentration and use it to determine the unknown protein concentrations. If the sample is available as a precipitate, then dissolve the precipitate in 2N NaOH.
Peterson has described a precipitation step that allows the separation of the protein sample from interfering substances and also consequently concentrates the protein sample, allowing the determination of proteins in dilute solution. Peterson's precipitation step is as follows: Vortex, and stand at room temperature for 10 min.
Decant the supernatant and then dissolve the pellet in 2N NaOH. The reaction is very pH-dependent, and it is therefore important to maintain the pH between 10 and Take care, therefore, when analyzing samples that are in strong buffer outside this range.
The incubation period is not critical and can vary from 10 min to several hours without affecting the final absorbance.
The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques.
The Lowry assay () is an often-cited general use protein assay. For some time it was the method of choice for accurate protein determination for cell fractions, chromatography fractions, enzyme preparations, and so on.
Protein Determination. The protein concentration of the samples is determined either by Lowry's method (27) or by using the BCA protein assay reagent (Pierce, Rockford, IL). Bovine serum albumin is used as standard for both methods, and the standard sample contains the same concentration of Triton X and buffer as the test samples. The reaction is similar to the well-documented Lowry assay. Quick Start™ Bradford Protein Assay The Quick Start Bradford assay kit is a simple one step procedure. with no need to dilute standards for determining the concentration of protein in a solution.
The “Lowry Assay: Protein by Folin Reaction” (Lowry et al., ) has been the most widely used method to estimate the amount of proteins (already in solution or easily-soluble in dilute alkali) in. 1. OBJECTIVE To standardize a procedure for the determination of protein by modified Lowry at Sigma-Aldrich, St. Louis, MO. 2. SCOPE This procedure applies to all products that have a specification for Lowry protein determination without Trichloroacetic Acid (TCA) precipitation.