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Biuret Protein Assay.
(USING STEROX0 at 3l0 nm
Range: 0.07mg (A 0.1) to 0.5mb (A 0.7)
REAGENT
There are two reagents to use: Biuret A and Biuret B. Biuret B is made by diluting Biuret A with dH20. Therefore when using Biuret B use 1ml/1.5ml assay volume and when using Biuret A use.150ml/1.5ml assay volume. Biuret A is useful when large volumes are needed because of low protein concentration. Biuret A is also used by those who are concerned about the reagent that remains in pipette tips. The Pipettman manufacturer does not guarantee good delivery of solutions which tend to cling to the surface of the pipette tip. They recommend drawing up those types of solutions without pre-rinsing the tip, then wiping the outside of the tip, releasing the solution into the assay tube, rinsing the tip with the contents of the tube, vortex, rinsing again, and vortex. With the smaller amount of reagent the P200 can be used which can be inserted into the assay tubes much easier, and the tips are cheaper. It is believed that with the more dilute reagent, Biuret B, this problem should be minimal. Basically which reagent you use depends on personal preference and which campfire stories you’ve heard.
Biuret A/45 = Biuret: 30% NaOH, 2% STEROX = 1:4
.50 ml are added to each assay tube (total volume = 1.5 ml
Biuret B/5 - Biuret A/5: dH20 =.15:1
1 ml is added to each assay tube (total volume = 1.5 ml
Preparation of Reagents:
A. First make Biuret 1.5% CuSo4 , 6% NaK Tartarate; 30% Na OH (w/v); 2% STEROX (v/v).
To make 250ml dye reagent:
1)3.75g CuSo4 in approx. 100ml H20 in a beaker;
2)15.0g NaK Tartarate in approx. 100ml in another beaker;
3)Mix 1) & 2) in a 500ml beaker. Add and dissolve 75g NaOH (solid) and 5mk
STEROX.
4)Transfer to 250 ml grad. Cylinder or vol. Flask and add H20 to 250 ml. Store at room
temp, discard if a precipitate forms.
B. Dilute to make Biuret A/5.
To make 100 ml:
1)Make 30% NaOH by dissolving 30g NaOH in 70 ml H20 and bringing up to 100ml
with H20 in graduated cylinder.
2)Dilute Biuret x 0.2 with 30% NaOH. (E.g., 80ml NaOH = 20ml Biuret).
3)Add STEROX to 2% (e.g., 100ml + 2ml STEROX)
C. Dilute to make Biuret B/5.
To make 100ml:
1)15ml Biuret A diluted to 100ml with dH20.
STANDARD: Bovine Serum Albumin (BSA) from Sigma (catalog # ), ~20mg/ml in water,. The exact concentration Cst (mg/ml) = (A280-A320) / 0.66.
Procedure:
1)Prepare tubes (12 x 75mm is easiest to use when working with Wiretrols). Prepare 2 blanks, 3 standards (do in duplicate), and at least 2 tubes per unknown.
2)Plan assay by filling out form B310A.Frm or B310B.Frm (Biuret A @ 310nm or Biuret B @ 310 nm.).
a)Determine volumes of unknown to add. Optimum absorbance is 0.4, corresponding to about 0.3mg. If you don't know the approximate protein concentration then prepare duplicates with a couple of different amounts of protein (factors of 2 differences are useful).
b)Determine amount of buffer to add. The buffer that your sample is in should be added to each of the tubes containing the protein standard (BSA) if the buffer is known to affect the assay. MB and RB’s should not have nay effect, but to be on the safe side it is advisable to add them because of any contaminants which could affect the assay. It is absolutely necessary to add SB. The volume of buffer added to the standards should equal the maximum volume of protein solution assayed.
c)Determine amount of dH20 to add. The volume of dH20 is adjusted so that the total volume in each assay tube is 1.5ml, e.g.
vol. dH20 = 1.5ml ml reagent ml pro ml buffer
where ml reagent =.150 when using Biuret A and 1.0 when using Biuret B
ml pro = the ml of the unknown or the ml of BSA added
ml buffer = the maximum volume of protein added
= the volume of buffer added to the standards
= 0 for the tubes containing the unknown
3)Prepare tubes by adding ingredients from left to right as they appear on the assay forms, vortex after each addition except for the protein. Add protein with either wiretrols or drummond. But use the same instrument for standards as for the unknown. After reagent has been added it is safe to vortex protein solutions without clumping.
4)15 after adding the reagent balance the spectrophotometer at 310nm with the blanks. Read the tubes in the order that they were mixed. Rinse cuvette between samples (not necessary between duplicates).
5)Plot absorbance vs. mg protein for the standards. You may use the blanks as data points (0 absorbance at 0 mg). The plot should be linear through all the standards and through the 0,0 point. If the data cannot be plotted or shown on a screen so that bad points or deviations from linearity can be detected then calculate K = A310/mg from standard data. K should be about 0.7. If all standards give approximately the same value of K, then you’re OK. If not discard bad points. Higher values of protein may give higher K values due to saturation of reagent. This nonlinear range of absorbance should be avoided. The alternative is to construct a detailed standard curve, which is not worth it.
6)Calculate the [pro] of the unknown(s) from the slope of absorbance vs. mg standards added as follows:
Abs
slope = -----
mg
mg/ml of unknown = Abs of unknown 1
------* ------
slope ml of unknown added
Instead of using a computer slope the average of K can be used.
Note
- Be sure to vortex after each addition except protein
- Run duplicate tubes.
- If need to add more sample, make sure total volumes are the same in all tubes.
- The undiluted Biuret reagent can be used in the range of 0.50mg (A540 0.1) to 5 mg (A540 1), following the same procedure.
Biuret B/5
310nm
Data Sheet
DATE OF ASSAY: ______
Duplicate assays are run for each row below.
Additions are in the order indicated, from left to right.
Total volume is 1.5ml.
Read absorbance at 310nm.
See Biuret method for details.
BSA standard used: Date=______, cst (mg/ml = A280/0.66)=______.
Sample / H20ml / buff
ml / BSA
ml / Sample
ml / Rgt
ml / A
310 / A
310 /
310 / K
mg/A
Blank / / / 1.000 / / 0 / 0 / 0 /
ST1 / 0.010 / / 1.000 /
ST2 / 0.020 / / 1.000 /
ST3 / 0.030 / / 1.000 /
/ / 1.000 /
/ / 1.000 /
/ / 1.000 /
/ / 1.000 /
/ / 1.000 /
/ / 1.000 /
/ / 1.000 /
/ / 1.000 /
/ / 1.000 /
/ / 1.000 /
Calculations:
slope of Abs vs. mg pro = ______r2 = ______
ave. K = ______SD = ______SEM = ______
Solutions for Biuret Assay.
A.Biuret Solution (1.5% CuSo4 , 6% NaK Tartarate; 30% Na OH (w/v); 2% sterox (v/v).
To make 250ml:
1)3.75g CuSo4 in approx. 100ml dH20 in a beaker;
2)15.0g NaK Tartarate in approx. 100ml in another beaker;
3)Mix 1) & 2) in a 500ml beaker. Add 75g solid NaOH and 5ml a STEROX.
4)Transfer to a graduated cylinder and adjust the volume to 250 ml with dH20.
B.Biuret A (20% Biuret Solution: 80% of 30% NaOH, 2% sterox):
To make 100 ml:
1)Make 30% NaOH by dissolving 30g NaOH in 100 ml dH20 (final volume).
2)Dilute 20ml Biuret Solution (1.5% CuSo4; 6% NaK tartarate; 30% NaOH 2% sterox) with 80ml of 30% NaOH (or any 1:4 dilution). Add sterox to 2% (i.e., add 2ml sterox to 100ml solution).
3)Store at room temp.
C.Biuret B/5:
To make 100ml:
1)15ml Biuret A plus 85% dH20.
2)Store at room temp.
Fajerlab under EDITMethods.doc10/20/1999 4:55 PM10/20/1999 4:55 PM