1. The document describes a protein purification procedure using ammonium sulfate precipitation and the Folin-Lowry assay to measure protein concentrations. Bovine serum albumin was used as the standard protein.
2. The Folin-Lowry assay involves two reactions - first with an alkaline copper tartrate solution and then with Folin-Ciocalteu reagent. This allows spectrophotometric measurement of protein concentrations.
3. Ammonium sulfate precipitation was used to isolate proteins based on their solubility. This increased the yield and purity of the target protein, progesterone receptors.
Text Version is Available at: http://www.biochemden.in/2014/11/anthrone-method-carbohydrate-determination.html
Carbohydrates are very important component of Storage and structural materials in the plants. The carbohydrates are stored as free sugars and polysaccharides. The basic units of carbohydrates are Monosaccharides. When hydrolyse the carbohydrates, gives monosaccharides, but when hydrolyse monosaccharides it can not be split into more simpler sugars. The hydrolysed product of Polysaccharide are estimating by the resultant monosaccharides.
การวิเคราะห์ฤทธิ์ต้านอนุมูลอิสระที่ระยะต่างกันในกล้วยเล็บมือนาง
Analysis of Antioxidant Activity at Different Stage in Musa (AA group) ‘Kluai Leb Mu Nang’
อดิศร จำรูญ
Text Version is Available at: http://www.biochemden.in/2014/11/anthrone-method-carbohydrate-determination.html
Carbohydrates are very important component of Storage and structural materials in the plants. The carbohydrates are stored as free sugars and polysaccharides. The basic units of carbohydrates are Monosaccharides. When hydrolyse the carbohydrates, gives monosaccharides, but when hydrolyse monosaccharides it can not be split into more simpler sugars. The hydrolysed product of Polysaccharide are estimating by the resultant monosaccharides.
การวิเคราะห์ฤทธิ์ต้านอนุมูลอิสระที่ระยะต่างกันในกล้วยเล็บมือนาง
Analysis of Antioxidant Activity at Different Stage in Musa (AA group) ‘Kluai Leb Mu Nang’
อดิศร จำรูญ
Kinetic Spectrophotometric Determination of Drugs Based On Oxidation by Alkal...IOSR Journals
Simple, accurate and precise spectrophotometic methods for quantitative determination of four drugs viz., Levofloxacin (LEV), Moxifloxacin (MOX), Pseudo Ephidrine (PSE), Torsemide (TOR) have been developed based on oxidation of the drugs by alk.KMnO4. Kinetics of the oxidation reaction is followed spectrophotometrically, as one of the reaction product, Mn(VI), absorbed at 610 nm. Initial rate and fixed time method are used for the construction of calibration curves Beer’s law is obeyed in the range 6.25-37.5 μg ml-1 for LEV; 5-30 μg ml-1 for MOX; 6.25-37.5 μg ml-1 for PSE and 2.5-15 μg ml-1 for TOR. Recovery studies using pure samples and pharmulations in the Beer’s Law limits have been carried out. Excellent recoveries indicate the methods are accurate and precise. The methods have been validated in terms of ICH guidelines. Statistical analysis in terms of student’s t- test and variance F- tests demonstrate high accuracy and precision and suggest the methods can be applied in bulk drug and pharmaceutical industries.
Haemolysis effect of Mefenamic Acid 250 mg Capsule in Bio analysis by liquid ...IOSR Journals
A rapid, simple and specific method for estimation of Mefenamic acid in human plasma was validated using Indomethacin as internal standard. The analyte and internal standard were extracted from plasma using simple solid phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mM Ammonium Acetate in Water and acetonitrile (20:80, v/v) and detected by tandem mass spectrometry in negative ion mode. The ion transition recorded in multiple reaction monitoring mode were m/z 240.1 196.0 for Mefenamic acid and m/z 356.1312.0 for internal standard. Linearity in plasma was observed over the concentration range 35.000 – 7000.000 ng/mL for Mefenamic acid. The cv of the assay was 4.89 % to 5.98 % and accuracy was 99.36 to 102.20 % Intra and Interday respectively at LLOQ level. The validated method was applied to bioequivalence study of 250 mg Mefenamic acid in 28 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range
B. Pharm. (Honours) Part-III Practical, Pharmacology II,MANIKImran Nur Manik
a) Estimation of blood glucose by enzymatic method.
b) Estimation of blood glucose by chemical method.
c) Estimation of aspirin after oral administration by UV spectrophotometric method.
d) Estimation of aspirin after oral administration by calorimetric method.
e) Estimation of plasma protein by enzymatic method.
f) Estimation of plasma protein by burette method.
g) Estimation of blood uric acid level by enzymatic method.
h) Estimation of Paracetamol after oral administration by UV/Visible spectrophotometric method.
i) Handling of experimental animals: mice and rat.
j) Different routes of administration of drugs in experimental animals.
k) Assay of serum SGOT and SGPT activities in mice.
l) Assay of serum alkaline phosphatase activity
m) Isolation and determination of cholesterol content of biological samples.
VALIDATED LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY METHOD FOR DETERMINA...Manik Ghosh
A simple, highly sensitive and rapid LC-MS/MS method has been developed and validated for the quantification of metolazone in rat plasma using irbesartan as internal standard (IS). After simple protein precipitation extraction by acetonitrile, the analyte and IS were extracted from 50 μL plasma sample on an Agilent Poroshell 120, EC- C18 (50 mm × 4.6 mm, i.d., 2.7 μm) column using 5μL injection volume with a total run time of 2 min. Acidified methanol/water mixture was used as a mobile phase. The parent/product ion transitions for metolazone (m/z 366.1/258.9) and IS (m/z 429.2/207.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was found to be linear in the range of 0.05 – 200 metolazone. The method was validated with respect to selectivity, linearity, accuracy, precision, recovery and stability according to accepted regulatory guidelines. The described method was successfully applied to preclinical pharmacokinetic studies of analytes after an oral administration of metolazone (1 mg/kg) in rats.
Spectrophotometric methods for determoination of Proteins Sabahat Ali
Different types of assay are present of determination of proteins
Bicinchoninic acid assay, Biuret protein assay, Lowry test assay, Bradford protein assay & Warburg Christion (A280/A260)
The automation of sample preparation has become an increasingly important component for reproducible and operator-independent experiments. This work outlines novel strategies that are being utilized for automated online and offline sample preparation to achieve specific goals, such as a host of applications including targeting post-translationally modified proteins, non-tryptic peptides, and intact proteins.
the presentation contain ways used to estimate proteins, this presentation prepared by TONNYBITE, a student from KILIMANJARO CHRISTIAN MEDICAL UNIVERSITY COLLEGE, TANZANIA
COMPARATIVE INVESTIGATION OF FOOD SUPPLEMENTS
CONTAINING ASCORBIC ACID
Danka Obreshkova and Boyka Tsvetkova
Medical University – Sofia, Faculty of Pharmacy, Dept. of Pharmaceutical chemistry
Abstract. A simple, specific, precise and accurate reversed phase liquid chromatographic (RP-LC) method
has been developed for the determination of ascorbic acid in different food additives. The chromatographic
separation was achieved on a LiChrosorb C18, 250 mm x 4.6 mm, 5 μm column at a detector wavelength
of 230 nm and a flow rate of 1.5 ml/min. The mobile phase was composed of acetonitrile and water (60:40
v/v). The retention time of analyte was 3.49 min. The method was validated for the parameters like specificity,
linearity, precision, accuracy, limit of quantitation and limit of detection. The method was found to be
specific as no other peaks of impurities and excipients were observed. The square of correlation coefficient
(R2) was 0.9997 while relative standard deviations were found to be <2.0%. The proposed RP-LC method
can be applied for the routine analysis of commercially available food additives of ascorbic acid.
Kinetic Spectrophotometric Determination of Drugs Based On Oxidation by Alkal...IOSR Journals
Simple, accurate and precise spectrophotometic methods for quantitative determination of four drugs viz., Levofloxacin (LEV), Moxifloxacin (MOX), Pseudo Ephidrine (PSE), Torsemide (TOR) have been developed based on oxidation of the drugs by alk.KMnO4. Kinetics of the oxidation reaction is followed spectrophotometrically, as one of the reaction product, Mn(VI), absorbed at 610 nm. Initial rate and fixed time method are used for the construction of calibration curves Beer’s law is obeyed in the range 6.25-37.5 μg ml-1 for LEV; 5-30 μg ml-1 for MOX; 6.25-37.5 μg ml-1 for PSE and 2.5-15 μg ml-1 for TOR. Recovery studies using pure samples and pharmulations in the Beer’s Law limits have been carried out. Excellent recoveries indicate the methods are accurate and precise. The methods have been validated in terms of ICH guidelines. Statistical analysis in terms of student’s t- test and variance F- tests demonstrate high accuracy and precision and suggest the methods can be applied in bulk drug and pharmaceutical industries.
Haemolysis effect of Mefenamic Acid 250 mg Capsule in Bio analysis by liquid ...IOSR Journals
A rapid, simple and specific method for estimation of Mefenamic acid in human plasma was validated using Indomethacin as internal standard. The analyte and internal standard were extracted from plasma using simple solid phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mM Ammonium Acetate in Water and acetonitrile (20:80, v/v) and detected by tandem mass spectrometry in negative ion mode. The ion transition recorded in multiple reaction monitoring mode were m/z 240.1 196.0 for Mefenamic acid and m/z 356.1312.0 for internal standard. Linearity in plasma was observed over the concentration range 35.000 – 7000.000 ng/mL for Mefenamic acid. The cv of the assay was 4.89 % to 5.98 % and accuracy was 99.36 to 102.20 % Intra and Interday respectively at LLOQ level. The validated method was applied to bioequivalence study of 250 mg Mefenamic acid in 28 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range
B. Pharm. (Honours) Part-III Practical, Pharmacology II,MANIKImran Nur Manik
a) Estimation of blood glucose by enzymatic method.
b) Estimation of blood glucose by chemical method.
c) Estimation of aspirin after oral administration by UV spectrophotometric method.
d) Estimation of aspirin after oral administration by calorimetric method.
e) Estimation of plasma protein by enzymatic method.
f) Estimation of plasma protein by burette method.
g) Estimation of blood uric acid level by enzymatic method.
h) Estimation of Paracetamol after oral administration by UV/Visible spectrophotometric method.
i) Handling of experimental animals: mice and rat.
j) Different routes of administration of drugs in experimental animals.
k) Assay of serum SGOT and SGPT activities in mice.
l) Assay of serum alkaline phosphatase activity
m) Isolation and determination of cholesterol content of biological samples.
VALIDATED LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY METHOD FOR DETERMINA...Manik Ghosh
A simple, highly sensitive and rapid LC-MS/MS method has been developed and validated for the quantification of metolazone in rat plasma using irbesartan as internal standard (IS). After simple protein precipitation extraction by acetonitrile, the analyte and IS were extracted from 50 μL plasma sample on an Agilent Poroshell 120, EC- C18 (50 mm × 4.6 mm, i.d., 2.7 μm) column using 5μL injection volume with a total run time of 2 min. Acidified methanol/water mixture was used as a mobile phase. The parent/product ion transitions for metolazone (m/z 366.1/258.9) and IS (m/z 429.2/207.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was found to be linear in the range of 0.05 – 200 metolazone. The method was validated with respect to selectivity, linearity, accuracy, precision, recovery and stability according to accepted regulatory guidelines. The described method was successfully applied to preclinical pharmacokinetic studies of analytes after an oral administration of metolazone (1 mg/kg) in rats.
Spectrophotometric methods for determoination of Proteins Sabahat Ali
Different types of assay are present of determination of proteins
Bicinchoninic acid assay, Biuret protein assay, Lowry test assay, Bradford protein assay & Warburg Christion (A280/A260)
The automation of sample preparation has become an increasingly important component for reproducible and operator-independent experiments. This work outlines novel strategies that are being utilized for automated online and offline sample preparation to achieve specific goals, such as a host of applications including targeting post-translationally modified proteins, non-tryptic peptides, and intact proteins.
the presentation contain ways used to estimate proteins, this presentation prepared by TONNYBITE, a student from KILIMANJARO CHRISTIAN MEDICAL UNIVERSITY COLLEGE, TANZANIA
COMPARATIVE INVESTIGATION OF FOOD SUPPLEMENTS
CONTAINING ASCORBIC ACID
Danka Obreshkova and Boyka Tsvetkova
Medical University – Sofia, Faculty of Pharmacy, Dept. of Pharmaceutical chemistry
Abstract. A simple, specific, precise and accurate reversed phase liquid chromatographic (RP-LC) method
has been developed for the determination of ascorbic acid in different food additives. The chromatographic
separation was achieved on a LiChrosorb C18, 250 mm x 4.6 mm, 5 μm column at a detector wavelength
of 230 nm and a flow rate of 1.5 ml/min. The mobile phase was composed of acetonitrile and water (60:40
v/v). The retention time of analyte was 3.49 min. The method was validated for the parameters like specificity,
linearity, precision, accuracy, limit of quantitation and limit of detection. The method was found to be
specific as no other peaks of impurities and excipients were observed. The square of correlation coefficient
(R2) was 0.9997 while relative standard deviations were found to be <2.0%. The proposed RP-LC method
can be applied for the routine analysis of commercially available food additives of ascorbic acid.
Il programma del corso ICEP 2015 - International Course Endovascular Procedures, tenutosi a Roma dal 19 al 21 ottobre. Tra gli interventi anche quello della dottoressa Marcella Marletta che si è soffermata sul tema del costo dei dispositivi utilizzati nelle procedure endovascolari.
it is just a basic of image modification by matlab..
first save ur pic which u want to modify in a folder.. then go in matlab and type in command windown ......... imread(''COPY YOUR FILE LOCATION")........ and then start editing tyour pics using matlab
Marcella Marletta - Programma VIII Congresso Nazionale SihtaMarcella Marletta
Il programma dell'ottava edizione del Congresso Nazionale Sihta, tenutosi a Roma dal 1° al 3 ottobre, con la partecipazione, tra gli altri, della dottoressa Marcella Marletta.
La presentazione tenuta dalla dottoressa Marcella Marletta a Pisa, il 23 maggio del 2014, sul tema: "Costi e sostenibilità della Radiologia Diagnostica e Interventistica nella Sanità Pubblica".
Glycolysis (from glycose, an older term for glucose + -lysis degradation) is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+.
This presentation has detailed information on glycolysis. each step is explained in detail. there are certain videos which i have taken from youtube. if these videos are not viewable u can refer to shomus biology glycolysis videos. u will get a detailed info there.
lehninger 3rd edition is also very good for the structures
Happy studying :)
Escozine for Pets™ has 4 major production steps.
1. Collection of Scorpions from the Scorpion Reservation. 2. Extraction of venom, purification and therapeutic dose preparation. 3. Polarization of extract and quality control of Polarization 4. Manufacturing, quality control, warehouse and shipment.
Determination of Total Protein Using the LAMBDA UV/Vis SpectrophotometerPerkinElmer, Inc.
The Lowry and Biuret methods are standard methods for protein quantification. Though the latter is more sensitive and is used for investigative work, it is limited by (1) poor stability of the combined reagent, (2) non-reproducibility of color, especially at low protein concentrations, and (3) a non-linear chromogenic response with protein concentration. Ohnishi and Barr1 modified and simplified the Biuret combined reagent for the Lowry procedure and at the same time improved its stability. This application note describes the modified Lowry procedure for protein analysis.
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
Enzymatic Saccharification of Lignocellulosic BiomassBiorefineryEPC™
Enzymatic Saccharification of Lignocellulosic Biomass
YOU AGREE TO INDEMNIFY BiorefineryEPCTM , AND ITS AFFILIATES, OFFICERS, AGENTS, AND EMPLOYEES AGAINST ANY CLAIM OR DEMAND, INCLUDING REASONABLE ATTORNEYS' FEES, RELATED TO YOUR USE, RELIANCE, OR ADOPTION OF THE DATA FOR ANY PURPOSE WHATSOEVER. THE DATA ARE PROVIDED BY BiorefineryEPCTM "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE EXPRESSLY DISCLAIMED. IN NO EVENT SHALL BiorefineryEPCTM BE LIABLE FOR ANY SPECIAL, INDIRECT OR CONSEQUENTIAL DAMAGES OR ANY DAMAGES WHATSOEVER, INCLUDING BUT NOT LIMITED TO CLAIMS ASSOCIATED WITH THE LOSS OF DATA OR PROFITS, WHICH MAY RESULT FROM ANY ACTION IN CONTRACT, NEGLIGENCE OR OTHER TORTIOUS CLAIM THAT ARISES OUT OF OR IN CONNECTION WITH THE USE OR PERFORMANCE OF THE DATA.
Analysis of Aflatoxins in Pet Food by UHPLC Using PDA and Fluorescence DetectionPerkinElmer, Inc.
Commercially prepared pet foods are easy and economical ways to fulfill the nutritional requirements for pets. Dry pet food is produced with grains and cereal by-products rejected for human consumption. The contamination of these by-products, with toxigenic fungal metabolites called mycotoxins, pose a serious health threat to pets.
Aflatoxins, some of the most carcinogenic mycotoxins known, are classified as B1, B2, G1, and G2. Several aflatoxin outbreaks in commercial pet foods have been reported in the past few years. Symptoms from aflatoxin exposure include lethargy, anorexia, jaundice, and intravascular coagulation, the severity often varying based upon a pet’s breed, species, age, dose, length of exposure, and nutritional status. Even if affecting only a small percentage of commercial pet foods, problems with pet food safety impact the entire pet food industry due to recalls and loss of consumer loyalty. Such experiences have reaffirmed the need for commercial pet food manufacturers to devote extensive resources documenting product quality.
Clinical chemistry uses chemical processes to measure levels of chemical components in the blood. It is very useful for the early diagnostic of disease and for monitoring organ function. The most common specimens used in clinical chemistry are blood and urine. Table 1 shows the common blood tests and measurable items using UV/Vis spectrophotometers.In this application note, the cholesterol level in human serum was determined by the enzymatic method using the LAMBDA™ 465 UV/Vis Spectrophotometer and UV Lab™ software.
1. Protein purification-Folin-Lowry assay
Introduction
There were two main aims to the purification; the first was to calculate the total protein concentration
of the cytoplasm fraction before and after ammonium sulphate precipitation, and the second was to
calculate the specific activity of progesterone
The goal of the investigation performed is protein purification which is a series of processes used to
isolate a single type of protein from a more complex mixture within an organ, tissue or cell. These
processes are fundamental for discovering the structure, function, characteristics and interactions of the
protein being investigated. The protein used was standard bovine albumin. At the end of the
purification the protein will be separated from the non protein and all other proteins.
There are many other procedures used in order to measure the concentration of the protein in solutions.
The most widely used is the Folin-Lowry protein assay which was employed in this investigation to
find the protein content in the standard bovine serum albumin and of the unknown.
The procedure depends upon two reactions. The first uses the buiret reagent, which contains potassium
hydroxide(KOH) and copper II sulphate(CuSO4)along with potassium sodium
tartarate(KNaC4H4O6.4H2O) when in the presence of a protein the blue reagent turns violet.
The colour change is caused by alkaline solutions when copper sulphate complex containing Cu2+
ions
form reduced Cu+
ions with the reduced amide bonds of the proteins. Due to the nature of the assay
showing poor sensitivity, a second reaction follows the first. This involves using Folin-Ciocalteu
reagent, which measures the amount of protein needed in order to inhibit the oxidation reagent. It is
composed of phosphomolybate and phosphotungstate. The reduced copper is used to reduce the Folin-
Ciocalteu reagent by tyrosine and tryptophan residues, which have become attached to copper. The
reduced reagent is blue in color hence detectable with a spectrophotometer set in the absorbance range
of 500 nm to 750nm. The reagent increases the sensitivity by approximately a hundred times, for
detecting reduced copper. The assay is only linear over a range of protein concentration from 0µg/ml to
200µg/ml.
Ammonium sulphate precipitation is a method that is considered to make proteins precipitate, or “salt
out” of solutions, which are collected by centrifugation. The protein is solvated, or surrounded by
water, meaning that their solubility can be redecreased at high levels of salt. This is due to salt
behaving as an interference and attracting the water to itself. As a result the protein can “salt out”.
However salt concentrations differ from protein to protein and in order for it to salt out therefore
requiring centrifugation. The process precipitates all proteins that have the same hydrophobicity but
purifies progesterone receptors with a high yield of 70% to 80% with a 20-30 fold.
Method
Materials
For this experiment the following equipment was used;
• Test tube rack
• Test tubes(x16)
• Spectrophotometer
• Gilson pipette-P1000
• Gilson pipette-P5000
• Cuvette
• Labels
• Beaker
• Stopwatch
The reagents used throughout the experiment were;
2. • Standard bovine serum albumin(200 µg/ml)- 10ml
• Solution A, Copper Sulphate (1%w/v)- 1ml
• Solution B, Potassium sodium tartarate(2%w/v)-1ml
• Solution C, Sodium Carbonate (2% in NaOH)- 100 ml
• Folin-Ciocalteu reagent-6 ml
• Unknown 1- 1ml
• Unknown 2- 1 ml
• Deionised water
Procedure
Firstly, the spectrophotometer was switched on and set to a absorbance of 755 nm and let to warm up.
Next, it was necessary to prepare a series of protein standard solutions in duplicate from 0-200µg/ml by
pipetting the required amount of bovine serum albumin standard into a test tube and making up the
volume to 1ml with water.
The two unknown solutions (1 and 2) provided then had to be diluted by a factor of 10 and a factor of
100. From the two diluted unknowns (1+2), 1ml of each of the diluted solutions (in duplicate) was
placed alongside the standard solutions.
Then the 1ml of each of the solutions (A) Copper sulphate (1%w/v) and (B) Potassium sodium tartrate
(2%w/v) were mixed in with 100ml of solution (C) Sodium carbonate (2%w/v in NaOH, 0.1M). Once
theses solutions were mixed, 5ml of this was added to each of the protein standards and the unknowns
at a 30 second intervals.
Afterwards, it was necessary to dilute the Folin-Ciocalteu reagent (6ml) by a factor of 2. Once the
solution was diluted, it was required to add 0.5ml of this solution to each of the test tubes at exactly 10
mins after the previous solution was added. Then the tubes were well shaken and left to stand for
exactly 30mins.
Finally the spectrophotometer was then used to read the optical density of each solution against one of
the blanks. The results obtained were then used to plot a standard graph of protein concentration
against absorbance. From the graph it was now possible to determine the protein concentrations of
Unknown 1 and Unknown 2.
Results
This section illustrates the data obtained or calculated throughout the investigation.
At the start of the experiment protein standards were produced by dilution of standard bovine serum
albumin stock solution with de ionised water. Table 1 shows the amount of deionised water used to
dilute a specific volume of the stock solution in order to achieve the relevnt concentrations, with a total
volume of 1mL each time.
Table 1
For each protein standard solution, and for each of the unknown solutions
STD
solutions(µg/ml)
0 40 80 120 160 200
STD volume(ml) 0 0.2 0.4 0.6 0.8 1
Volume of water
added(ml)
1 0.8 0.6 0.4 0.2 0
3. At their various concentrations, the spectrophotometer was used to determine their absorbance’s after
centrifugation shown below in table 2. the absorbance’s were determined for the duplicates, and an
average absorbance calculated.
Concentratio
n (µg/ml)
0 40 80 120 160 200 U1x10 U1x10
0
U2x1
0
U2x10
0
1 0 0.153 .277 .323 .433 .
459
1.857 .714 .360 .08
2 .001 .140 .268 .329 .413 .
461
1.949 .689 .358 .082
Average
absorbance
0.00
5
0.146
5
0.272
5
0.32
6
0.42
3
0.4
6
1.901
5
0.7015 0.359 0.81
Table 2.
The values from table 2 were used to draw a calibration graph with the use of Microsoft Excel. The
graph is shown below
A calibration graph to show the relationship between the average concentrations of each standard
protein solution agaiinst absorbance
y = 0.0023x + 0.0466
R
2
= 0.962
0
0.1
0.2
0.3
0.4
0.5
0.6
0 50 100 150 200 250
concentration(ug/ml)
absorbance
Using the equation of the line y=0.0023x+0.0466. The concentration of the unknowns
could be found to be and respectively. This was found using the following equation
(for unknown 1)
4. Discussion
Considering the results obtained, there is a positive linear relationship between the
absorbance as the concentration of protein standard increases. Also the R value shows
the degree of error is low. Anomalies in the results may have been caused by
contamination of samples while pippetting. To reduce error and increase reliability of
results the experiments could be done in triplicate.
The purification is considered useful if it causes an increase in the specific activity of
the protein to be purified