Protein Measurement with the Folin Phenol Reagent
Journal of Biological Chemistry, 1951 · paper · Biomedicine
Describes procedures for measuring protein with the Folin phenol reagent, after studying how pH, reaction time, reagent concentrations and interfering substances affect it. The methods can determine as little as 0.2 microgram of protein.
Checked: PDF from developmentalbiology.wustl.edu, fetched 4 Oct 2026. Page numbers are those of that PDF.
1 finding
Substance Microanalysis procedure: "To the precipitate are added 5 μl. of 8 N NaOH" - 8 N conflicts with the macro procedure it scales down ("0.1 ml. of 1 N NaOH" before 1 ml of Reagent D), with footnote 2 ("The use of stronger alkali than 1 N did not appear to be an improvement") and with Fig. 3, where colour is near zero at 0.2 N NaOH.
- p. 3 Microanalysis, second paragraph
To the precipitate are added 5 μl. of 8 N NaOH
- p. 2 Insoluble Proteins, etc., second paragraph
0.1 ml. of 1 N NaOH to 5 to 100 γ of precipitated protein
- p. 3 Footnote 2
The use of stronger alkali than 1 N did not appear to be an improvement.
- p. 7 Fig. 3, x axis (NaOH normality) and curves
NaOH con- centration is calculated before addition of 0.1 volume of diluted Folin reagent
Only the authors can say what is right here.
Judge's note: Page 3 image and crop C002-j read clearly "5 μl. of 8 N NaOH" (the text layer garbles μl, but the 8 is printed). The micro volumes (5 μl NaOH, 50 μl Reagent D, 5 μl Reagent E) are the macro precipitate procedure (0.1 ml, 1 ml, 0.1 ml) divided by 20, and the text says the only necessary change is slender tubes. With 2 μl of 5% TCA left (about 0.6 μeq), 8 N gives about 0.69 N NaOH before the Folin reagent; 1 N gives about 0.08 N, matching the macro 0.09 N. Fig. 3 (page 7) shows the 1.6% Na2CO3 curve peaking near 0.08-0.1 N and falling to about 0.01 optical density at 0.2 N. Reagent D contains no acid and the Folin reagent (5 μeq) cannot absorb the excess. I found no statement in the paper explaining a stronger alkali for the micro scale. The conflict is real; whether 8 N is a misprint (for 1 N) or a deliberate unexplained choice only the authors can say, hence author_query.
Second check: another model agreed
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6 minor (typos and the like; most checked by the judge only)
Wording "values based on N determination would be overestimated by 15 to 20 per cent" - Table III gives N-based whole-tissue values 13 to 24 per cent above the extracted precipitate (brain 11.8 vs 9.5, skeletal muscle 20.1 vs 17.8).
- p. 8 Specificity and Interfering Substances, first paragraph
would be overestimated by 15 to 20 per cent
- p. 8 Table III, Brain, Based on N X 6.25, Extracted ppt. and Whole tissue
11.8
- p. 8 Table III, Skeletal muscle, Based on N X 6.25, Whole tissue
20.1
- p. 8 Specificity and Interfering Substances, first paragraph
be overestimated by 15 to 20 per cent
Judge's note: Read Table III on the page 8 image. N x 6.25 whole tissue over extracted precipitate: brain +24.2%, kidney +14.4%, liver +16.4%, muscle +12.9%, heart +17.7%. Expressed as a fraction of the whole-tissue value the range is 11.4 to 19.5%, also not 15 to 20. The companion "3 to 6 per cent" for Folin colour does fit (3.1 to 5.6%). No definition elsewhere changes the comparison base. The stated range is a loose summary: two of five tissues fall outside it, the direction and rough size are right, so this is a wording-level imprecision rather than a wrong result. This cluster also carried the skeletal-muscle summation point (18.8 vs 17.8 + 0.15 + 0.09); that is adjudicated under C006.
Second check: another model agreed
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Wording Table IV, right half, row "0.292 | 236 | 1.03 | 1.00": E(1%, 1 cm) of 236 does not follow from optical density 0.292 at 1.00 γ present in 0.082 ml (calculated 239), while the adjacent rows 0.280 -> 229 and 0.283 -> 233 do follow within about 1 unit.
- p. 9 Table IV, right half, second row (protein present 1.00)
0.292
- p. 9 Table IV, right half, first and third rows (protein present 1.00)
0.283
Only the authors can say what is right here.
Judge's note: Crop C007-a confirms the digits. Across the table E equals optical density divided by the concentration from the Present column (e.g. 0.095 at 0.33 γ gives 236.1, printed 236; 0.365 at 1.32 γ gives 226.7, printed 227). For the three 1.00 γ rows this gives 229.6, 239.4 and 232.1 against printed 229, 236 and 233. Rounding of the shared Present amount moves all three together and cannot fix one row; the ratio 236/229 = 1.031 is below 0.292/0.280 = 1.043 even at the rounding limits. The Found value 1.03 agrees with the optical density 0.292, so the E value is the likelier slip. The gap is about 1.4%, in a table where other rows are off by up to 1.5 units, so it is minor and the conclusion is unaffected.
Second check: another model agreed
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Wording Table IV, right half: rows "0.365 227 1.30 1.32" and "0.365 227 1.32 1.32" give two different Found amounts (1.30 and 1.32 γ) for the same optical density and E value; the row between, 0.367, gives 1.31.
- p. 9 Table IV, right half, 4th data row
0.365 227 1.30 1.32
- p. 9 Table IV, right half, 6th data row
0.365 227 1.32 1.32
Judge's note: Crop C007-a (whole of Table IV) confirms both rows as printed. Elsewhere in the table 0.001 of optical density corresponds to roughly 0.004 γ, so rounding of the optical density cannot produce a 0.02 γ spread, and 0.367 -> 1.31 sits between two different values for 0.365. The paper says only that Found is read against standards run under the same conditions; separate standards or blanks per replicate are possible but not stated. The size is 0.02 γ, the level the text itself calls the usual error, so this is a small consistency point with no effect on the conclusion.
Second check: another model agreed
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Wording Table IV, last two rows: "0.443 220 1.62 1.66" and "0.444 221 1.61 1.66" - the higher optical density is paired with the lower Found amount.
- p. 9 Table IV, right half, 8th data row
0.443 220 1.62 1.66
- p. 9 Table IV, right half, 9th data row
0.444 221 1.61 1.66
Judge's note: Crop C007-a confirms the digits. Found should rise with optical density, and rounding preserves order, so 1.62 and 1.61 look transposed unless the replicates were read against different standards or blanks (not stated). The difference is 0.01 γ, well inside the stated error, so this is a trivial consistency point.
Second check: another model agreed
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Typo Introduction: "has considerable merit for certain application, but that its peculiarities and limitations need to be understood" - singular "application" where the plural "applications" is required.
- p. 1 Introduction, third paragraph
has considerable merit for certain application
Judge's note: Crop C001-a shows "certain application," with no trailing s; the text layer agrees. Not an extraction artefact. Cosmetic.
Second check: none (judge only)
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Typo Summary, point 3: "The differences in the amount of color obtained with a number of proteins is recorded." - plural subject "differences" with singular verb "is" (should be "are recorded").
- p. 10 Summary, point 3
The differences in the amount of color obtained with a number of proteins is recorded.
Judge's note: Page 10 image shows the sentence exactly as quoted. Subject-verb disagreement; cosmetic.
Second check: none (judge only)