Reference no: EM132798352

PHAR2004 Renal and Endocrine Diseases - University of Nottingham

INSERT a labelled image of theSDS PAGE gel provided here:

Protein concentration (Bradford assay):

Complete the data table below:

Sample	Measured absorbance	Average absorbance
Blank 1
Blank 2
Blank 3
Unknown sample (2-fold dilution) replicate 1
Unknown sample (2-fold dilution) replicate 2
Unknown sample (2-fold dilution) replicate 3
Unknown sample (4-fold dilution) replicate 1
Unknown sample (4-fold dilution) replicate 2
Unknown sample (4-fold dilution) replicate 3
Standard 1 mg/ml
Standard 1 mg/ml
Standard 1 mg/ml
Standard 0.5 mg/ml
Standard 0.5 mg/ml
Standard 0.5 mg/ml
Standard 0.25 mg/ml
Standard 0.25 mg/ml
Standard 0.25 mg/ml
Standard 0.125 mg/ml
Standard 0.125 mg/ml
Standard 0.125 mg/ml
Standard 0.0625 mg/ml
Standard 0.0625 mg/ml
Standard 0.0625 mg/ml
Standard 0.0313 mg/ml
Standard 0.0313 mg/ml
Standard 0.0313 mg/ml

INSERT your BSA standard curve (imported from e.g. Excel):

Include R2 value for standard curve =
Linear regression (in the form Y = a + bX):

What is the original concentration of unknown sample? (show your workings):

SECTION 2
This section tests your understanding of what you have learned in the virtual practical and in lectures (and your logic) to answer the following questions:

Question 1

Olaide has just started a research project to study Steroid Receptor proteins. Her supervisor (Dr.Jones) gave her samples oftwo purified steroid receptor proteins from the lab freezer and asked her tocheck their purity by SDS PAGE. He left her a note with the following information:

After staining her gel, Olaide was able to see single bandsin each sample (Lanes C and D). Lane A contained the MW markers. Using the information provided above, she was able to work out which two of the five Nuclear Receptors Dr. Jones had given her.

a) Calculate the approximate molecular weights (expressed in kilodaltons) of each of the five receptors (to 1 decimal place).

b) Which nuclear receptors are most likely present in Lanes C and D

c) Identify which molecules below will bind to the receptors in C & D.

Question 2

James, a PhD student, is studying a human enzyme that is composed of two polypeptides. He bought the recombinant enzyme preparation from a commercial supplier and used SDS PAGE to check his protein. James set up two SDS PAGE gels (to replicate his experiment), but for Gel 2 he was in a rush to finish and forgot to add ?-mercaptoethanol to his protein sample.

a) Explain why this error led to the different results observed in Gel 1 and Gel 2. Comment on the size of bands obtained in each. (Max 50 words)

b) Can you suggest an amino acid that must be present in both subunits to account for the above observations?

c) Can any conclusion be made about the tertiary structure of this enzyme? (Max 50 words)

Question 3.
Dr Patel is using bacterial expression to purify a 115 amino acid recombinant polypeptide (known as KAP2). The KAP2 protein can be purified by binding it to an affinity column. He set up a 1 litre culture of E.colicells expressing recombinant KAP2. The cells were harvested, lysed and the lysate applied to the affinity purification column. After washing to remove other proteins, a buffer solution was applied which causes KAP2 to elute from the column, and 1 ml fractions were collected. Dr.Patelthen used 20μl each from fractions 5-12 for analysis by standard SDS PAGE gel. The final gel stained with Coomassie Blue is shown below. Dr.Patel also performed a Bradford assay on fractions 6-12 (he discarded fraction 5 due to low protein content) and calculated how much protein (μg) was present in each 20μl sample.

a) calculate the molecular weight of KAP2 in kilodaltons (to 2 decimal places)

b) Which two fractions contain the highest TOTAL amount of KAP2
Explain your answer (Max 50 words)

c) Which 3 fractions contain the most highly purifiedKAP2?
Explain your answer (Max 50 words)

d) If Dr Meyer combines the 3 most highly purified 1 ml fractions, what is the total yield of purified KAP2 he obtained from 1 litre of culture?
e) What is the percentage yield of pure KAP2 within the eluted fractions ?
Express your answer to 1 d.p.

Question 4
Johas started studying a transcription factorprotein (ZXR2). Jo has analysed ZXR2 by SDS PAGE and observed that is approximately 50kDa. ZXR2 shares 95% sequence homology with the related protein ZXR1, which is known to bind a specific 6 bp DNA sequence found near the promoters of its target genes.
Jo wondered if ZXR2 might also bind this sequence, and he came up with the following experiment to test his hypothesis. Jo mixedZXR2 protein with an equal amount of a100 base pair double stranded DNA fragment that containsthree copies of the ZXR1 binding sequence. After incubating the DNA and protein together for 1 hour, he added an equal volume of 2X loadingbuffer, boiled the sample for 5 minutesand performed standard SDS-PAGE, followed by Coomassie Blue staining.

a) Describe how many bands you would expect to be visible on the gel and their molecular size?

b) What effect will adding DNA have on the mobility of ZXR2? Will the DNA fragment be visible? Explain your answer (Max 50 words)

Question 5

Hannah is studying the Coronavirus SARS-CoV-2 spike protein. She has used recombinant DNA techniques to clone a short region of the Spike protein called the receptor binding motif (RBM). This is the region that binds the human ACE2 cell surface receptor . She inserted the RBM sequence into an expression vector, and hopes to purify this from cultured cells for in vitro studies. The sequence of Hannah's construct is shown below.

a) What are the additional Sequences A & B that Hannah engineered into her construct, and why have might they been added? (maximum 50 words)

b) The two highlighted amino acids in the RBM have been found to be important for ACE2 binding and are mutated in a new UK Coronavirus variant as shown below. Hannah wants to change her construct by PCR site-directed mutagenesis to study this variant. Suggest single base changes in both codons that would enable her to generate the new variant sequence below.

VEGFYCNFPLQ

Note: SECTION 2 from q1

Attachment:- Renal and Endocrine Diseases.rar