Description
I’m working on a chemistry multi-part question and need the explanation and answer to help me learn.Hello I have completed part 1 which you will see and need you to complete part 2 which are tables 5,6,7 and the discussion. I will provide all the results from the lab and an example video on how to do the calculations. I have left comments in red on what every column requires. All is attached below, please do not disrupt part one only complete part 2. Please be knowledgeable in biochemistry calculations and enzyme purification before accepting. Please be careful of units. I NEED AS SOON AS POSSIBLE I NEED IT NOW PLESE
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Lab Report – Purification and Activity of Alkaline Phosphatase Enzyme
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Everything in red is part of the template, should be addressed in report and deleted.
As I go through and complete things I’ll move them from the pages to attached notes.
Additional note: For some reason when I transferred the template it converted every “μg”
to mg. I think I got them all but if you see mg anywhere that doesn’t make sense thats
why
Captions ABOVE for tables BELOW for graphs
Abstract 20%)?
3. What stage of purification did you lose the most alkaline phosphatase? What procedure do
you think caused this (and why)?
4. What stage of purification did you see the greatest purification of alkaline phosphatase?
What procedure do you think caused this (and why)?
Reference:
Ninfa, A.J., Ballou, D.P. and Benore, M., 2009. Fundamental laboratory approaches for
biochemistry and biotechnology. John Wiley & Sons.
HZ.
Fundamental Laboratory Approaches for Biochemistry and Biotechnology
113
Chapter 4: Quantification of Protein Concentration
Protein + Cu2+
OH-
Cu 1+
LOWRY METHOD OF PROTEIN DETERIVHNATION
The Lowry method is one of the most commonly used methods of
protein determination because it is inexpensive, easy to perform, very
sensitive, and highly reproducible. Indeed, the publication describing the
method (Lowry et al., 1951) is one of the most highly cited publications
in the history of biochemical research. As of the time of preparation of
-ooc
coo-
-ooc
coo-
Cu1+ + 2BCA
this edition of this book, this manuscript has been cited by more than
80,000 other papers, according to the Google Scholar (Chapter 15).
Nevertheless, the assay has certain serious limitations: it is sensitive to a
variety of contaminants, the standard curves are oniy linear at low
protein concentrations, and the timing and mixing of reagents with the
samples must be precise. It is essential to create a standard curve each
time protein determinations are done.
The Lowry reaction consists of the Biuret reaction (described
V.
above), followed by the reduction under alkaline conditions of the FolinCiocalteu reagent (phosphomolybdotungstate mixed acids). Copper ions
facilitate the reduction process. The principle chromogenic groups are
the peptide linkages in complex with copper (Biuret), and the blue
reduced molybdotungstates, which are largely reduced by Tyr, Trp, and
15
polar amino acids. Therefore, the sensitivity of the test depends upon the
composition of the protein. The product of the reaction,
heteropolymolybdenum blue, is intense blue with an absorption
maximum of ~ 750 nm. This wavelength is usually out of the range of
interfering colors.
BICINCHONINIC ACID (BCA) IV1ETHOD FOR PROTEIN
DETERMINATION
Ta
Figure 4-2 BCA reaction
DYE-BINDING METHOD (BRADFORD METHOD) OF PROTEIN
DETERMINATION
The binding of the dye, Coomassie® Brilliant Blue G-250 (Figure 4-3),
to proteins causes a shift in the absorption maximum of the dye from
465 nm (red) to 595 nm (blue) in acidic solutions (Bradford, 1976;
Sedmark and Grossberg, 1977). This dye forms strong, noncovalent
complexes with proteins via electrostatic interactions with amino groups
and carboxyl groups, and by van der Waals forces. Since the color
response does not maintain linearity over a wide range of protein
concentration, it is strongly recommended that a standard curve be run
with each assay. The dye is prepared as a stock solution in phosphoric
acid. The method is a simple one-step procedure in which the dye
reagent is added to the samples and the absorbance is measured at 595
nm. The method is quite sensitive and accurate, but the reagent stains
The reaction is similar to the Lowry reaction, except that bicinchoninic
cuvettes and is somewhat difficult to remove. Dye-binding protein
acid (BCA) is used in place of the Folin-Ciocalteu reagent (Figure 4-2).
assays are compatible with most common buffers, chaotropic reagents
Cu2+ is reduced to Cul+ by protein in alkaline solution (the Biuret
such as 6 M guanidine-HCl, and 8 M urea, and preservatives such as
reaction). Two molecules of BCA chelate to a cuprous ion resulting in
sodium azide. However, high concentrations of detergents can interfere
an intense purple color with an absorbance maximum at 560 nm. The
with this assay. A very similar dye, Coomassie Brilliant Blue R-250 is
sensitivity of the method is similar to the Lowry method (20-2000
used to stain protein bands in gel electrophoresis (Chapter 6).
^g/mL of the unknown sample), but it is not as sensitive to certain
In addition to the Coomassie Brilliant Blue G-250 binding assay
contaminants such as detergents. However, the reaction is more sensitive
(Bradford method), a variety of other dye binding assays have been
to interference from carbohydrates than is the Lowry reaction. The BCA
developed or are under development. These include assays based on
method is not a true end point method, because the color continues to
different dyes, such as Bromocresol Green (Spencer et al., 1977),
develop slowly over time. However, after an incubation of 30 min at 37
Pyrogallol Red (Watanabe et al., 1986), and Eosine Y (Hong et al.,
°C, the color is sufficiently stable for reliable measurements (~2.5% drift
1999). Binding of these dyes (or molybdenum complexes with these
per 10 min). Some of the substances reported to interfere with the BCA
dyes) to proteins is sensitive to a variety of contaminants and displays
method are: catecholamines, tryptophan, lipids, phenol red, cysteine,
variable degrees of specificity, as in the Bradford method, and thus these
tyrosine, impure sucrose or glycerol, H202, uric acid, and iron. Why do
assays may hold an advantage in particular circumstances (Durgawale
you think these substances interfere with the assay? ^
etal.,2005).
±N_
Fundamental Laboratory Approaches for Biochemistry and Biotechnology
>r 4: Quantification of Protein Concentration
115
Watanabe, M., Kamei, S., Ohkubo, A., Yamanaka M., Ohsawa, S., &
Tokuda, K. (1986). Urinary protein as measured with a pyrogallol
red-molybdate complex, manually and in a Hitachi 726 automated
so;
SOg
analyzer. Clin. Chim. 32:1551-54.
OCgHs
OBJECTIVES
Figure 4-3 Coomassie Brilliant Blue G-250
REFERENCES
You will be provided with a protein standard (BSA) at a known
concentration (10 mg/mL) and several samples containing protein at
unknown concentrations. Some of these samples may contain pure BSA,
Bradford, M. (1976). A rapid and sensitive method for the quantitation
while others may contain different proteins or mixtures of different
of microgram quantities of protein utilizing the principle of protein
proteins. In addition, some of the samples may be spiked with
dye-binding. Anal. Biochem. 72:248-54.
sulfhydryl-containing compounds, detergents, carbohydrates, etc., which
will interfere with one or more of the assays. Your objective will be to
Durgawale, P., S. Kanase, Shukla, P. S., & Sontakke, S. (2005). A
sensitive and economical modified method for estimation of
12;
cerebrospinal fluid proteins. Indian Journal of Clinical Biochemistry
20:174-77.
Gornall, A. G., Bardawill, C. J., & David, M. M. (1949). Determination
measure protein concentration by the Biuret, Lowry, BCA, and Bradford
methods for each of these samples. By comparing the results from these
experiments, you may be able to deduce which samples were protein
mixtures, and which samples contained interfering substances.
of Serum Proteins by means of the Biuret Reaction. J. Biol. Chem.
177:751-66.
BSA standard and construct a BSA standard curve for each assay
Hong, H.-Y, Yoo, G. S., & Choi, J. K. (1999). An eosine Y method for
method. Some general guidelines for this are that for the Biuret and
The first step in the analysis will be to prepare dilutions of the
protein determination in solution. Analytical Letters 32:2427-42.
Lowry methods, you will use a series of samples with different known
Lowry 0. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951).
amounts of protein, that are 0.5 mL each. For the BCA and Bradford
Protein measurement with the Folin phenol reagent. J. Biol. Chem.
193: 265-75.
methods, you will use a series of samples for the standard curve that are
Pierce Chemical Co., Instructions for Coomassie® Protein Assay
much higher than the amount used in the other assays, as this assay is
Reagent # 23200.
0.1 mL each. The amounts of protein used in the Biuret assay should be
not as sensitive as the others. Be sure to have a good number of points
Peterson, G. L. (1979). Review of the Folin phenol quantitation method
within the sensitivity range of each assay method (see below). To
of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100:201-
determine the apparent protein concentrations in the unknowns, you
20.
should examine several dilutions of each. This step is necessary to
Sedmark, J. J., & Grossberg S. E. (1977). A rapid, sensitive and versatile
ensure that at least one of the dilutions used results in a value falling
assay for protein using Coomassie brilliant blue G250. Anal.
Biochem. 79:544-52.
within the linear portion of the standard curve. In addition to the
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F.
is good experimental design to perform the assays on the standards and
H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J.,
unknowns side-by-side at the same time. This insures that variables such
& Klenk, D. C. (1985). Measurement of protein using bicinchoninic
as temperature, time, etc. are internally controlled. In addition, it is
acid. Ana/. Biochem. 150:76-85.
useful to perform the assays in duplicate, so that large pipetting errors
undiluted samples, 3-fold and 10-fold dilutions are reasonable guesses. It
Spencer, K., & Price, C. P. (1977). Influence of reagent quality &
may be readily recognized and experimental error can be ascertained.
reaction condition on the determination of serum albumin by
We will discuss the results obtained by the different methods and
bromocresol green dye binding method. Ann. Clin. Biochem. 14:105-
compare results from different groups using the different techniques.
15.
This will give us some real results to better understand the differences
between precision, accuracy, and reproducibility.
Fable 3: B
118
Fundamental Laboratory Approaches for Biochemistry and Biotechnolo
122!2a^.
iter 4: Quantification of Protein Concentration
we
119
SapnpN
Test Tj
PROCEDURE
-Test^
Make up a set of standards using BSA or some other protein
“Test
source in the range of 5-2000 ^g/mL. In the BCA procedure,
Test
you will use 0.1 mL of each sample.
Copper sulfate: Sigma C6283, 250 g
Test
To 0.1 mL of your unknown samples (and dilutions of your
Sodium potassium tartrate: Sigma S6170, 500 g
samples), and to 0.1 mL of the standards add 3 mL of BCA
working reagent into labeled test tubes. Mix well. Incubate
Potassium iodide: Sigma P2963, 100 g
tubes at 37 °C for 30 min. The unknowns and the standards
Sodium carbonate: Sigma S7795, 1 kg
should be measured in the same experiment.
Cool to room temperature and read the absorbance at 560 nm
3.
4.
Sodium hydroxide: Sigma S0899, 500 g
against a blank of sample buffer processed as above.
2N Folin-Ciocalteu reagent: Sigma F9252, 500 mL
Determine your protein concentrations) from a standard curve.
BCA (4, 4′-Dicarboxy-2, 2′-biquinoline): Sigma D8284, 10 g
Sodium bicarbonate: Sigma S6297, 1 kg
12
Note: Instead of preparing the BCA solution, this reagent
may be purchased Sigma or Pierce (see below). In that
case, BCA and sodium bicarbonate will not be required.
MATERIALS
Dye reagent: Use vigorous homogenization or agitation to
dissolve 100 mg of Coomassie® Brilliant Blue G-250 in 50 mL
of 95% ethanol. This solution is mixed with 100 mL of 85%
phosphoric acid, diluted with water to 1 liter, and filtered. The
reagent is stable for 2 weeks at room temperature (The mixed
95% ethanol
Phosphoric acid
Reagent or from BioRad as BioRad Protein Assay mixture.
Note: Instead of preparing the dye reagent, this reagent
may be purchased from Sigma, Pierce, or BioRad (see
below). In that case, Coomassie Brilliant Blue G-250, 95%
ethanol, and phosphoric acid will not be required.
Make up a set of standards using BSA or some other protein
source in the range of 5-2000 pig/mL. In the Bradford procedure,
you will use 0.1 mL of each sample.
To 0.1 mL of your unknown samples (and dilutions of your
samples), and to 0.1 mL of the standards add 3 mL of Bradford
reagent. Mix well and let stand at room temperature for 5 min.
The unknowns and the standards should be measured in the same
experiment.
Measure the absorbance at 595 nm against a blank consisting of
100 uL of sample buffer or water and 3 mL of the dye reagent.
4.
Coomassie Brilliant Blue G-250: Sigma B5113, 100 mg/L
working solution
reagent can be purchased from Pierce as Coomassie® Protein
PROCEDURE
3.
BCA pre-mixed solution: Sigma B6943 or Pierce 23225
Determine your protein concentration(s) from a standard curve.
Bradford reagent (pre-mixed): Sigma B6916, Pierce 23200,
or BioRad 500-0006
Purchase answer to see full
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