΢χολή Μηχανολόγων Μηχανικών ΕΜΠ
Διδάςκων: Λεωνίδασ Αλεξόπουλοσ
Αρχζσ Βιολογικήσ Μηχανικήσ
3ο ΜΑΘΗΜΑ: ΕΡΓΑ΢ΣΗΡΙΟ
Πόςα γραμμάρια πρωτεΐνησ περιζχει ζνα κφτταρο;
STEPS (the big picture)
Step 1: You play around with pipettes and you learn how to handle liquids
Step 2: You prepare liquids with known concentrations of protein
Step3: You mix those liquids (in a predetermined ratio) with a chemical (BCA)
that binds to all proteins and gives a product that absorbs at 562nm. The more
the protein, the more the product, the higher the absorbance. Thus, you create
a “standard curve” where you know the absorbance for each protein
concentration.
Step 4: You mix (at the same ratio) your unknown liquid with the same
chemical. You measure absorbance and with the standard curve you calculate
how much protein concentration you should have in the liquid.
Step 5:Your task - Write a short report (max. 1 page)including the following
1. A photo of the 96 well plate before and after the absorbance
measurement
2. The standard curve and the fitting of the unknown samples
3. You need to find out, (i) the concentration and the total amount of
protein on this vial, (ii) the total amount of protein (of the 47million
cells), and (iii) how much protein each cell contains
You have lysed 47millions cells in 12ml buffer (whatever liquid) and you now have a vial with 50ul.
Once you know the concentration, you multiple by the volume to get the total amount of protein and then
divide by the number of cells to calculate the protein per cell.
You write the report and you send it to: [email protected]
Deadline: 2 weeks after the lab
General Guidelines
Making Solutions
1. Mass concentration (MASS IN LIQUID): mass/volume [i.e. mg/ml, 5ug/ml ]
The mass concentration is defined as the mass of a solid constituent divided by the volume of the mixture.
Example: A “stimuli” (a protein that triggers response) can be potent at 100ng/ml. In order to prepare the
stimuli we will weight 100ng and dilute them in 1ml (in practice, we will get a small amount or protein,
weight it, find out the weight in μg and then add the corresponding liquid)
2. MOLARITY: Molar concentration (moles per liter, = 1Μ or molar).
1M solution would consist of ΜW in grams in one liter aqueous dissolvent
Example: Gefitinib has MW:446. How many ug do I need to get 1uM Gefitinib in 1L?
3. Volume concentration (v/v) (LIQUID IN LIQUID): [90%]
Is defined as the volume of a LIQUID constituent divided by the volume of the mixture. Example: We need
70% ethanol. We add 70ml ethanol + 30ml H2O
4. Weight in volume (w/v) (SOLID IN LIQUID): [1%]
A one percent solution is defined as 1 gram of solute per 100 milliliters final volume.
Examples:
Weight in volume: Prepare 2 liters 0.85% sodium chloride (NaCl) αλάτι
Molarity: Prepare 200 ml of 70 mM sucrose (ζάχαρη)(C12H22O11, MW:342)
Two hundred milliliters is 0.2L and 70 mM is 0.07M. The molecular weight of sucrose can be determined from its
chemical formula, namely C12H22O11 and the atomic weights of carbon, hydrogen, and oxygen. The formula weight for
sucrose is identical to its molecular weight, namely 342.3 grams per mole. A 1M solution would consist of 342.3
grams sucrose in one liter final volume. A concentration of 70 mM is the same as 0.07 moles per liter. Take 0.07
moles/liter times 342.3 grams per mole and you have 23.96 grams needed per liter. To make 200 milliliters of your
solution multiply grams/liter by liters needed. Since 200 milliliters is 0.2L, multiply 23.96 grams by 0.2L to get 4.792
grams needed. Since a typical top loading electronic balance displays mass to the nearest 0.01 gram, the amount to
be weighed should be rounded to 4.79 grams, although it is perfectly acceptable and perhaps even preferable to
round to 4.8 grams.
Prepare 50ml 30mM sucrose if your stock solution is 70 mM sucrose
C1*V1=C2*V2
Prepare 100ml 75% ethanol from 90% ethanol
BCA Protein Quantification Assay
BCA protein assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the
colorimetric detection and quantitation of total protein.
Protein concentrations generally are determined and reported with reference to standards of a common
protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared
from the protein and assayed alongside the unknown(s) before the concentration of each unknown is
determined based on the standard curve.
Preparation of BSA Standard Curve
Bovine serum albumin (also known as BSA or "Fraction V") is a serum albumin protein derived from cows. BSA is used
because of its ability to increase signal in assays, its lack of effect in many biochemical reactions, and its low cost,
since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.
Dilute solution of BSA 2 mg/mL in NaCl into clean vials using the same diluent as the sample(s) (in our case
lysis buffer – LB).
Preparation of BCA Working Reagent (WR)
Use the following formula to determine the total volume of WR required:
(#standards+#unknowns)x(#replicates)x(volume of WR per sample)=Total volume WR required
WR is prepared by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B).
For the microplate procedure sample to WR ratio is 1:8 for 20-25 μL of protein sample. If sample size is limited 10 μL
of each unknown sample and standard can be used but sample to WR ration is 1:20 and the working range of the
assay is limited to 125-2000 μg/mL.
Procedure
1.
2.
3.
4.
5.
6.
7.
Pipette desired volume of each standard and unknown sample replicate into a micoplate well e.g. 25 μL
Add appropriate volume of WR to each well e.g. 200 μL
Mix plate thoroughly on a plate shaker for 30 seconds
Cover plate and incubate at 37oC for 30 minutes
Cool plate at room temperature
Measure absorbance at or near 562 nm on a plate reader
Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562nm
measurements of all other individual standard and unknown sample.
8. Prepare a standard curve by plotting the average Blank-corrected 562nm measurement of each BSA
standard versus its concentration in μg/mL.
9. Use the standard curve to determine the protein concentration of each unknown sample.