Question

1. Figure I shows an SDS-PAGE gel. A) Rank the 3 proteins by size, from largest to smallest. Explain why this trend is observed in SDS-PAGE gels. B) What is the purpose of SDS in SDS-PAGE? C) Sample L is the ladder. What is its purpose? D) Typically, PA (polyacrylamide) is used as the gel for protein electrophoresis, whereas agarose is used for DNA electrophoresis. Explain why a different gel material is used, Specifically referring to the pore size of agarose vs. PA. Hint: Think about the size of DNA vs. proteins. Figure 1 Sample # 2 3 4 L 1 5 6 Protein 1 Protein 2 Direction of migration Protein 3 2. A) Calculate the extinction coefficient in units of M cm') of the 3 peptides below. You can use an online tool to do so; c.g. enter the sequence into https://web.expasy.org/protparam. i) GYRGDSPG ii) GWRGDSPG il) GORGDSPG B) Why are the extinction coefficients different for the 3 peptides? C) What would be the absorbance of a 1 mg/ml solution of peptide ii, if you use a spectrophotometer with a 1 cm path length? Use the Beer Lambert law, A-sel 3. You run a PCR reaction, starting with 100 copies of a template gene. How many PCR cycles will it take to generate two million copies of the gene, in the ideal scenario? How many cycles will it take if the PCR reaction is only 93% efficient? 4. The speed of DNA replication at a replication fork is about 100 nucleotides per second per stand in human cells. What is the minimum number of origins of replication that a human cell must have in

Answer 1

Question 1

1. Protein 3 is the smallest in size followed by Protein 2 and then Protein 1 being the largest in size. SDS-PAGE is a type of gel electrophoresis where a charged biomolecule is separated on the basis of charge to mass ration under a constant electric field. Considering same charge, small size biomolecules will move faster under the electric field due to their small size and thus have lee resistance to their movement. In other words, small size biomolecules will have high electrophoretic mobility as compared to large size biomolecules and due to high electrophoretic mobility, these small biomolecules will cover more distance in the direction of migration relative to their large size biomolecules. That’s why Protein 3 being the smallest one moves faster and occur a larger distance in the direction of migration followed by Protein 2 and then Protein 1 being the largest one covers less.

Hence, the ranking by size, from largest to smallest would be; Protein 1 the largest followed by Protein 2 and then the Protein 3, the smallest one.

From largest to smallest in size, Protein 3 > Protein 2 > Protein 1.

2. SDS stands for Sodium dodecyl sulfate. It is an anionic detergent. SDS is used to denature proteins converting multimeric proteins to dissociate into their subunits but the majorly it gives proteins similar charge to mass ration thus eliminating the factor of charge from the basis of separation. For example, there are two proteins Protein A and Protein B, if Protein A have a very high negative charge as well as in size as compared to B. Due to its high negative charge it will be more attracted to the positive charge during electrophoresis and thus it will migrate more compared to B and thus seems smaller in size yet in reality it is larger. To eliminate this SDS is used where it imparts negative charge by binding to every two amino acid residue and by this charge to mass ratio becomes similar between proteins and now, these proteins can be accurately separated out on the basis of size only.

3. Suppose an unknown band is there, how do we know it’s size in gel. The only way is comparing the band with calibrated one i.e. if we know 1Kb size band comes at this position of gel than by comparing our unknown band we can say whether our DNA band is 1Kb or not. This is the same thing DNA ladder do. It is used to determine the size of unknown DNA band by comparing with the ladder band and it’s given catalogue.

4. Usually agarose gel based electrophoresis is used to separate out very large biomolecules with a molecular mass greater than 200 kDa while polyacrylamide based electrophoresis can be used to separate out small size biomolecules also ranging from 10 kDa to 1000 kDa and as given in the hint usually the size of DNA is very large and enough t get very good resolution using agarose based but as protein sizes are usually small from 20-30 kDa to not so big. That’s why polyacrylamide is used as the gel for protein electrophoresis whereas agarose is used for DNA electrophoresis.

The given question is a type of question with multiple questions (question 1-2) and according to HomeworkLib guidelines under the given condition answer only the first question unless the student has not specified (which is not in the given case). Thus, I answered only the first question i.e. Question-1.