Lab 1: Spectrophotometry
Lab objectives
- become familiar with the use of spectrophotometer -create standard curves and determine the unknown concentration of 2 compounds - to determine the maximum wavelength (y-max) of Bovine Serum Albumin (BSA) using the Bradford and/or Coomassie Blue Method
P2, P20, P200, P1000
-0.2-2 -2-20 -20-200 -200-1000
P10, P100, P5000
-1-10 -10-100 -1000-5000
Your blank cuvette should *only* contain
0.1N NaOH
To pipette 50uL set to what?
050
Objectives of lab 1- Measurement, pipette use and the standard curve
1. Accurately and precisely use pipettes in a laboratory setting. 2. Dilute a concentrated stock solution to a working concentration. 3. Use a spectrophotometer 4. Generate and evaluate a standard curve, 5. Use a standard curve to determine the concentration of protein in milk.
how do you use a micropipettor
1. Choose the correct instrument 2. Adjust the dial to the correct volume. Turn it clockwise to decrease and counterclockwise to increase. 3. Select a tip 4. hold upright and press the first stop (half way) 5. Put in the liquid and release back to the rest position and remove the micropipettor. 6. Hold pipettor at a 10 45 degree angle against the inside wall of the receiving vessel to make sure there is no bubbles. 7. press to the first stop and then all the way to the 2nd stop to blow the rest of the liquid out. 8. return to the rest position 9. eject the tip
To pipette 100uL set to what?
100
Grey bottoms have ___ modes
2 modes, no need to switch between 0-1, 0-2 setting
Protein concentrations of milk
2% milk 33.3 ug/ul Skim milk 33.3 ug/ul Almond milk 4.17 ug/ul
Short cuvettes must be ___ full
2/3
Blue bottom spectrophotometers have ___ mode settings. Explain
3 0-1A = absorbance range -.3 - .999 0-2A= -.3 - 1.99 %T = sample transmission
Creating a standard curve for p-nitrophenol using dilutions of a stock solution: set your wavelength setting to __nm
400
what is the λ-max for p-nitrophenol?
400nm
Maximum absorbance wavelength of bovine serum albumin
595nm
equation for absorbance (include units)
A=abc or A=εbc A= absorbance a=absorptivity (litre/g cm) ε= molar absoptivity ( litre/ mole cm) b= path length (1 cm for nova spec) c= concentration (moles/litre or g/litre)
Concentration measured by spec
Absorbance divided by slope
BSA stands for
Bovin Serum albumin
One of the simplest but most useful spectrophotometric assays used in cell biology
Bradford and/or Coomassie Blue method
indirect assay also called
Bradford method and/or coormassie blue method
Part A of lab 1
Bradford reaction-Bradford reagent sodium, chloride and BSA. 1-Adjust the pipette to the volume of BSA 2-Pipette into the glass cuvette 3-Dispose of the tip and adjust the volume of sodium chloride 4- Add sodium chloride 5-Adjust again and add BSA last 6-Flick the tube 7-Leave the tube for 3-5 minutes and transfer to the cuvettes by pouring 8-Set the wavelength on the spectrophotometer and start with the blank. Always insert the cuvettes in the same direction and clean the outside with a chemical wipe. 9-Insert and close the lidAnd push the A one hundred T button. 10-Then begin with your samples
Calculation for table 1
C1 is the concentration you need to start with. This means that you need the stock concentration of the BSA protein. C2 is the final BSA protein concentration that you want in your solution. V2 is the final volume that you want your solution to be in. V1 is the volume of BSA protein you need to get the final concentration in the final solution. (X)
Concentration of unknown
C1V1=C2V2 Solving for C1
Lab 1 Learning Objectives
Correctly use micropipettes for volumetric analyses. Correctly use the spectrophotometer to measure absorbance. Construct a serial dilution and calculate the dilution factor achieved. Construct and use a standard curve to determine concentration of protein in milk.
Lab data analysis lab 1
Create a scatter plot with concentration on the X-axis and absorbance on the Y-axis. Add a line of best fit (this line represents the trend of the data set overall). Calculate the regression coefficient (R2) to statistically test how well the data fit the linear equation. The closer this number is to 1.0, the better the data fits the regression line. For this experiment, an R2>0.97 indicates acceptable precision and accuracy in your technique. Based on the standard curve, determine the concentration of your unknown sample. Compare this calculated concentration to the known concentration provided by your TA.
Calculate the concentration of the unknown using the dilution factor
Dilution factor equals total volume divided by initial volume. Multiply this value by whatever concentration you get
What happens to R Squared values if you exclude the last two values in the curve? does that change the concentrations of your unknown? If so which should you use to calculate the unknown concentration in milk?
If I exclude the last two values the R Squared value increases. This slightly changes the concentrations of my unknown because the slope changes which is used to calculate both types of concentration. I should use the higher R squared value because it gives my graph more accuracy and a better trendline to determine my R squared value and slope
To avoid having to wipe your cuvettes with ___ between each reaching, what can be done?
Kimwipe do not touch the middle region of the cuvette with your hands. Fingerprints will affect your readings.
blank
Prepare a blank of the same solution but without the analyte, having the same pH and similar ionic strength; a necessary step as the cell and solvent can scatter some light.
What was the name of the absorbance machine?
The Nova Spec
Look at the final two values of your standard curve on the scatter plot, do they correlate strongly with the rest of your data? Why or why not?
The final two values do not correlate strongly because the concentration is so low which in turn decreases the absorbency below the line of best fit
Part B of lab one Milk dilutions
The purpose is to measure the protein concentrations of different kinds of milk. 1-Make a standard curve 2-Create a serial dilution because the protein concentration in the milk samples are too high For this type of assay to handle. 3- Label three tubes for each of the milk samples. 1-10, 1-100, 1-1000 1-10=Nine parts water and one part milk. One 10th of the original concentration. 1-100=Nine parts water and one part of the 1/10 dilution. 1-1000=Nine parts water and one part of the 1/100 4- Create a Bradford assay Using 10 µL of each dilution. 5-Compare colors to the original standard curve and determine which is the best for your sample. The best sample is the one that isSimilar to the middle of the standard curve. 6- Take volumes from a table 3 7-Find the absorbances 8- Calculate the concentrations. Remember this sample is diluted so multiplied by whatever the dilution sample was.
Bradford reagent
Turns blue in the presence of proteins
Calculate concentration measured
Use the equation from your trendline and plug in the absorbency for the Y value and solve for X
Part B of lab 1
You will obtain a sample of milk from the TA and record the identity of the milk (what kind of milk is it?). Set up Bradford reactions as before but use the volumes of unknown listed in Table 3. (Follow steps 2-7 from Part A of Lab Activity 2, with the exception of the unknown volumes.) Use the same blank and wavelength as in Part A of Lab Activity 2. Measure and record the absorbance of each diluted milk sample in Table 3. Once your standard curve is created, you will be able to compare the absorbance of the diluted milk sample to the standard curve equation to determine the concentration of BSA (protein) in your milk.
Calculate the absorptivity (_) and molar absorptivity (_) of p-nitrophenol using what?
a ε absorbance reading and concentration from your graph
Standard curve
a graph in which properties of known samples are measured and plotted. The direct proportionality of these properties can then be used to determine the same information from unknown samples. In this lab, a standard curve of Absorbance versus protein concentration will be used to determine the concentration of protein in an unknown sample.
Each spectrophotometer includes
a light source, a collimator, which is a lens or focusing device that transmits an intense straight beam of light, a monochromator to separate the beam of light into its component wavelengths, and a wavelength selector, or slit, for selecting the desired wavelength. sample holder, a photoelectric detector, which detects the amount of photons that are absorbed, and a screen to display the output of the detector.
Accuracy
a measure of how close a result matches a correct or standard value. For example, how close a dart is to the bullseye in a dartboard is an indicator of accuracy.
Molar weight
a measure of mass. By definition, a mole is 6x1023 objects (atoms, molecules, Aggies, etc.). The mass of an object is defined by its composition at the atomic level.
Precision
a measure of variability. For example, three darts placed close together on a dartboard indicate high precision, but if this cluster of darts is far from the bullseye it would also indicate low accuracy.
Aliquot
a portion of something (noun); or to divide or transfer a portion of something (verb). For this lab, aliquot refers to an amount of liquid
multichannel pipet
a type of pipet that holds 4-16 tips from one plunger; allows several samples to be measured at the same time
Measure the ____ and ____ of all dilutions of p-nitrophenol, beginning with the lowest concentration.
absorabance %transmittance
Standard curve of p-nitrophenol had ___ on the y axis and ____(__) on the x axis
absorbance concentration mM (mmoles/mL)
Beer's law states that absorbance is directly proportional to
absorptivity, path-length, and concentration
Allow the sample to reach the appropriate temperature and mix it gently, so that bubbles are not introduced. The sample can them be
added directly to the cuvette, within the instrument, and a reading taken.
When you turn on machine, what do you do?
allow the machine to go through 3 calibration checks (90sec) in which the letters CAL are displayed. When calibration complete the display changes from CAL to 360.
Spectrophotometer
an instrument for measuring the intensity of light that is transmitted through a sample (transmittance) or absorbed by a sample (absorbance). In this lab, you will measure light absorbance at a defined wavelength. The use of a spectrophotometer is called spectrophotometry. ultraviolet and visible range can determine the concentration in the sample
Micropipette
an instrument used to measure/deliver amounts of liquid with high accuracy and precision. Micropipettes are expensive so treat these with respect.
Micro-volume spectrophometers
are optimal for measuring the quality and concentration of expensive samples of limited volume, such as biomolecules, including proteins and nucleic acids.
Both DNA and RNA have an absorbance maximum
at 260 nm from which their concentration can be determined. The purity of the nucleic acid can also be assessed from the ratio of absorbance readings at specific wavelengths.
When using long cuvettes, both the blank and sample cuvette must be
at least 1/2 full to obtain an accurate reading
Must have a ____ cuvette to check that the absorbance reading remains at "0" each time you take a reading.
blank
BSA was used for the ___ method
bradford (Coomassie blue)
One common application of the spectrophotometer is the measurement of
cell density. Cell density measurement is useful in generating logarithmic growth curves for bacteria, from which the optimal time for induction of recombinant protein can be determined.
When white light passes through a solution containing a coloured compound, what happens?
certain wavelengths of light are selectively absorbed
The spectrophotometer can also be used to measure
chemical reaction rates. In this example, absorbance is used to monitor an enzymatic reaction by the disappearance of a reaction intermediate at 452 nm over time. The rate of this enzymatic step can be calculated by fitting the data to the appropriate equation.
One of the most common uses of a spectrophotometer?
creating a standard curve and determining the unknown concentration of the particular compound.
Never forget to close the door as UV radiation emitted from an open spectrophotometer can
damage the eyes and skin
What does a spectrophotometer do?
detects the amount of radiant energy absorbed by molecules in a solution
What are standard curves useful for?
determining the unknown concentration of the particular compound
We used both of theres methods
direct assay, indirect assay
new spectrophotometers
directly coupled to a computer, where the experiment parameters can be controlled and results are displayed.
Recently, the introduction of micro-volume spectrophotometers has
eliminated the necessity for sample holders. Such spectrophotometers use surface tension to hold the sample.
Depending on the type of spectrophotometric experiment you are performing, it may be necessary to
generate a standard curve prior to sample measurement, from which the concentration of your sample analyte can eventually be determined.
Absorptivity of a compound is how much light the molecule can absorb at a given wavelength and concentration in ___ while molar absorptivity is when the concentration is in ___
grams per liter moles per liter
Bradford Assay
is an assay that uses Coomassie Blue dye to bind the common part of all proteins, that is the peptide backbone. When Coomassie binds to proteins, it changes from reddish to blue. This color change is easily detectable by a spectrophotometer allowing the concentration of protein to be measured quantitatively. By itself, Bradford reagent has a reddish-brown color and the highest absorbance (absorbance spectrum maximum) at 465 nm. Bradford reagent bound with proteins, however, is blue and has an absorbance spectrum maximum at 595 nm. As protein concentration increases, more of the protein-dye complex will be formed and the resulting solution will be a more intense blue color.
Transmittance
is the fraction of light that passes through the sample and is defined as the intensity of light passing through the sample over the intensity of incident light.
Absorbance
is the inverse logarithm of transmittance and is the quantity your spectrophotometer will measure.
Spectrophotometer with a blue bottom use
long cuvettes
beam of light
made of photons. When photons encounter molecules in the sample, the molecules may absorb some of them, reducing the number of photons in the beam of light and decreasing the intensity of the detected signal.
Determined the _____ of a compound and used this to create a _____
maximum wavelength standard curve
Lambda max was used to make the BSA standard curve and to determine the unknown quantity of BSA because
measurements at this value will give the smallest error
After the Coomassie blue dye was added, what do you do?
mix immediately with vortex mixer
What to do with a micropipettor
never turn above or below the indicated volume. change tip after each time. store in the upright position.
2 unknown concentrations where of
p-nitrophenol and BSA
direct assay also called
p-nitrophenol experiment
With constant ___ and ___, absorbance will increase interlay with respect to equal increases of concentration.
path-length wavelength
After performing the absorbance measurement on your sample,
proceed to the appropriate calculation for your experiment; for example determination of concentration or the rate of enzyme activity.
After wiping any fingerprints and spills off the outside of the cuvette,
properly insert the cuvette in the sample holder and close the door to the cuvette compartment.
An ideal experiment yields
results with both high accuracy and high precision. Although biological and/or environmental conditions may limit one or both of these features, poor technique is an unacceptable source of errors.
BSA-Coomassie blue method: At each change of wavelength, do what?
set reference using blank
Spectrophotometer with a grey bottom use
short cuvettes
Repeater pipet
small volume to a large amount
Bradford assay
spectroscopic analytical procedure used to measure the concentration of protein in a solution
Beer-Lambert Law
states that there is a linear relationship between the absorbance and concentration of a sample. concentration of the sample solution can be determined. absorbance is the product of the extinction coefficient, a measure of how strongly a solute absorbs light at a given wavelength, the length that light passes through the sample, or path length, and the concentration of solute.
If your spectrophotometer shows a blinking .999, then
switch to 0-2 mode (absorbance is greater than 1)
In order to measure a substance with a spectrophotometer,
the absorbance of the substance must fall within the range of wavelengths the spectrophotometer can measure. Most proteins, by themselves, do not have an absorbance within this range.
Place cuvette in the chamber making sure that
the line on the cuvette corresponds with the alignment dot in the chamber and that the lid is closed
What indicates what type of micropipettor you have
the number at the top of the plunger and the color
It is best if you use a reading of the standard curve from what portion ?
the straight portion
Traditional spectrophotometer sample holders are designed to
to hold plastic and quartz cuvettes.
Before measuring the UV-visible spectrum of a sample,
turn on the machine and allow the lamps and electronics to warm up
Applications of micropipettors
volume transfer loading samples- dna gel disrupting tissue
Tip color meanings
white .5-2.5 yellow 1-200 blue 200-1000
Since the blank contains everything except the p-nitrophenol, is it included in the graph?
yes
Set the desired wavelength or wavelength range to be transmitted at the sample, which depends on the optimal wavelength of light that the analyte absorbs. Then,
zero the instrument by taking a reading of the blank, which will subtract the background from your sample buffer.
