need some help with these problems. thanks for your help! 1.) In “Lab 2 – CellFr
ID: 51863 • Letter: N
Question
need some help with these problems. thanks for your help!
1.) In “Lab 2 – CellFract”, there were two high speed centrifugations. The first one, which led to the isolation of the mitochondrial fraction, was spun in the SS-34 rotor (radius of 10.7cm) at 11,000rpm. The second one, which led to the isolation of the microsomal and cytosolic fractions, was spun in the TLA-100 rotor (radius of 3.89cm) at 45,000rpm. The lab claims that these two centrifugations were spun at gravitational forces equal to 15,000 x g and 105,000 x g, respectively. Show the mathematical calculations for gravitational force and indicate whether the notes were correct or not.
2.) Bovine serum albumin (BSA) will be used in “Lab 3 – ProtQuant” as a protein standard. If BSA is a 66.5 kDa protein, what is the molarity of the 1mg/ml stock solution used in this lab?
BSA (µg)
Abs595
0
0.003
1
0.077
2
0.148
3
0.202
5
0.368
7
0.466
10
0.633
3. As part of a Bradford protein assay, you record the absorbance readings at 595nm of different amounts of BSA in order to create a protein standard curve. The table below indicates the average absorbance reading for each amount of BSA measured. Use Excel to plot the mass of BSA (x-axis) against the absorbance at 595nm (y-axis) in order to determine the equation for the linear trendline for this data. What is that equation?
4. You dilute your nuclear fraction 1:10 by mixing 50µl with 450µl water. You then mix 5µl of the diluted nuclear fraction with the Bradford reagent and record an absorbance of 0.412 at 595nm. Using the equation from the trendline in Question 3, determine the protein concentration of the original (undiluted) nuclear fraction in µg/ml?
5. If all of the samples collected from “Lab 2 – CellFract” (homogenate, nuclear, mitochondrial, microsomal, cytosolic) had been suspended in the same volume (e.g. 8ml), would likely have the highest protein concentration? Explain.
BSA (µg)
Abs595
0
0.003
1
0.077
2
0.148
3
0.202
5
0.368
7
0.466
10
0.633
Explanation / Answer
Question 1
Answer: mathematical calculation for gravitional force
G force refers to Relative Centrifugal Force (RCF). RCF is positively related with the rotor radius and the rotation speed of the centrifuge. The g force rpm conversion formula is as follows:
RCF = 1.118 * 10-5 * r * (rpm)2
Where:
RCF: Relative Centrifuge Force, in "g"
r : Rotor radius, in "cm"
rpm: Rotation speed, rotations per minute, in "rpm"
For mitochondrial fraction,
RCF = 1.118 x 10-5 x 10.7 x (11,000)2 = 14475 x g
For microsomal and cytosolic fractions,
RCF = 1.118 x 10-5 x 3.89 x (45,000)2 = 88067.655 x g
From the above calculations it is clear that the mentioned values or notes provided for "g" or RCF are not correct.
Question No 2.
Answer:
Question No 3.
Answer:
y - y1 = (y2 - y1 / x2 - x1) (x - x1)
Solve the equation for y.
In our case, y1 = 0.418 ; y2 = 0.466; x2 = 7; x1 = 2
So, equation comes out to be
y = 0.0636x + 0.0208
Answer: The highest amount of protein concentration will be in homogenate that contains a variety of membrane-enclosed organelles like nuclear, mitochondrial, microsomal and cytosolic.
Cells can be broken up in various ways: they can be subjected to osmotic shock or ultrasonic vibration, forced through a small orifice, or ground up in a blender. These procedures break many of the membranes of the cell (including theplasma membrane and membranes of the endoplasmic reticulum) into fragments that immediately reseal to form small closed vesicles. If carefully applied, however, the disruption procedures leave organelles such as nuclei, mitochondria, the Golgi apparatus, lysosomes, and peroxisomes largely intact. The suspension of cells is thereby reduced to a thick slurry (called a homogenate or extract) that contains a variety of membrane-enclosed organelles, each with a distinctive size, charge, and density.
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