10:24 .. Done 8259949 119590643 _Kafour In trying to determine whether the polyc
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10:24 .. Done 8259949 119590643 _Kafour In trying to determine whether the polycyclic aromatic hydrocarbon (PAH) Pyrene induces any DNA damage in bronchial epithelial cells it was hypothesized that elevated levels of the protein P53 may be an indicator ef DNA damage in BE cells Design your experiment with triplicates of 2 groups of BE cells (each group of 1x 10* cells): group 1 is a control group without any treatment with Pyrene, Group2: exposure (treatment) to pyrene at a concentration of 1 M for 1 hr At the termination of exposure, the cellular supernatant volume is 0.5 ml of each group of cells for which determination of total protein levels is required To determine if P53 is induced by Pyrene, a performed to separate proteins [in preparation of westerm blotting techmique-to be performed later, ie.you will perform separation of the proteins only at this stage. ssay was Show your calculations of the preparation of 10 HM of pyrene (prepared in 10 ml of Hank's Buffered Saline Solution with 0.3% DMSO). Sbow your calculations knowing that the volume of pyrene solution is 10 ml Molecular Mass of Pyrene is 202.25 g/mole (202g/mole) To separate proteins using gel-electrophoresis, it was determined that a loading of 20 ug of total protein would be required. Show your calculations of determining totalamount of protein in each group of cells (control group: group 1 and group 2: exposed to pyrene) Discuss the materials and methods used in performing gel-electrophoresis Good Lack and have funExplanation / Answer
Calculations for preparation of 10uM pyrene solution.
1. preparation of 0.3% DMSO in 10 ml hanks balanced salt solution
0.3 % of 10 ml = 0.3/100 x 10 = 0.03 ml = 30 microliters (1ml = 1000 microliters)
total volume is 10 ml = 10000ul
10000- 30 ul = 9970 ul
In 9970 ul hanks balanced salt solution add 30 ul DMSO.
2. Preparation of 10uM pyrene
Molarity = number of moles/ volume of solution in liters
number of moles = given mass/ molecular mass
= g / 202 = 0.0049 g ( let denote given mass with g)
volume of solution is 10 ml . converting it to litres:
1 L = 1000 ml
1 ml = 1/1000L
10 ml = 10/1000 L = 0.01 L
Molarity = g / 202 / 0.01 L
10 uM = 0.0049g / 0.01 L
10 x 10-6 = 0.0049 g / 0.01 L
10 x 10-6 X 0.01 = 0.0049 g
10-7 / 0.0049 = g
g = 0.0204 x 10-3 = 20.4 x 10-6 = 20.4 ug
20.4 ug pyrene should be added to 10 ml hanks balanced salt solution with 0.3 % DMSO
2 . total amount of protein in sample can be determined via bradford assay. Cells of each group should be lysed using RIPA ( RAdioimmunoprecipitation assay buffer) . Centrifugation is done after lysing to separate proteins into the supernatant. .
Series of dilution of standard BSA protein are made in a 96 well plate. Like 0.2 , 0.4, 0.6, 0.8 1 and 1.2 ug . dilutions of group 1 and group 2 samples are also made i.e 1:10 . Bradford reagent is added to each well and reading is taken at 595 nm.
A standard curve of absorbance vs concentration of dilutions of standard BSA protein is made. The absorbance of group 1 and group 2 samples is known via bradford assay. The protein concentration of unknown group 1 and group 2 sample is calculated by either by extrapolating or equation of the curve. Now the concentration calculated is of diluted unknown sample. The actual protein concentration of unknown sample is = concentration dervied via standard curve x dilution factor ( 10 in this case)
3. Material required for gel electrophoresis of protein are :
Acrylamide, Tris -HCL ( ph 8 and 6.8) , Sodium dodecyl sulphate ( SDS) , ammonium persulfate, TEMED, Commassie brilliant blue, Glycinate buffer containing tris base, glycine and SDS, cracking buffer containing Tris.cl, beta mercaptoethanol , SDS, glycerol and bromophenol blue.
SDS PAGE is a discontinuos gel. The top gel is called the stacking gel having large pores. The ph of this gel is 6.8 . A phenomenon of isotachophoresis occurs in this gel. Protein molecules are stacked between glycinate and chloride ions. Glycinate ions are behind the protein molecules and chloride ions are movng ahead of proteins. Ths helps in aligning all protein molecules so that they start migrating into the resolving gel from the same point.
Resolving gel has high ph of 8.8 . Its pores size is smaller which helps in separation of proteins according to molecular weight. As soon as the proteins, glycinate and chloride ion reach the resolving gel isotachophoresis stops and glycinate trails ahead of protein breaking the pattern .
step 1 : sample preparation
Protein sample are incubated with cracking dye at 100 degree C for 10 minutes. This steps helps in denaturation of proteins and proteins are separated on the basis of molecular weight only.
Step 2 : electrophoretic run
samples are loaded into wells and electrophresed at constant current of 30 milli ampere till bromophenol blue ( tracking dye) reaches the bottom of the gel
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