Below is an EMSA showing four different reactions, A-D. In each tube there is some combination of labelled DNA probe, Protein X (the protein you are studying), and an antibody for Protein X. Identify which combination of components are found in each of the four reactions and explain how you determined that based on the molecular interactions being studied and your knowledge of gel electrophoresis. It is possible that multiple lanes have the same component(s). A B C D EMSA

Monoclonal antibodies can be conjugated to an insoluble support by chemical methods. Explain how these antibody-bound beads can be exploited for protein purification.

A bloactlve hexapeptide was cleaved by trypsln from a larger proteln. The peptlde was sequenced by treatment with varlous reagents and enzymes. The results are as follows: a) Treatment wlth cyanOgen bromlde gave a tetrapeptlde of compositlon asp, trp, cys and met. b) Treatment with chymotrypsln gave a tripeptlde of compositlon asp, trp and cys, a dipeptlde wlth the same C-term and an amlno acld. c) Treatment with streptococcal protease gave a dipeptlde and a tetrapeptlde. The composltion of the peptlde Is lys, trp, asp, cys and met 1. What Is the sequence of the hexapeptlde? 2. What is the pl of thls peptlde?

Gel Electrophoresis A. Why does DNA travel toward the positive electrode in the gel chamber? B. What is a ladder sequence and why is it useful to include in a gel?

Various antimicrobial drugs to treat microbial infection have diverse mechanism of action. Consider the following antimicrobial drugs: A.    Seconeolitsine, known as DNA topoisomerase I inhibitor in bacteria. (i)              Explain briefly how inhibiting DNA topoisomerase I is a good mechanism of action for an antibiotic, include possible molecular machineries being targeted. (ii)            What would be an appropriate response if seconeolitsine works well by stating the state of supercoiling in bacteria. (iii)          To prove your answer (ii), you test the condition of bacterial DNA by running gel electrophoresis, one has been treated with seconeolitsine (+ sample) and the other one is not (- sample). Explain the position of each + sample and – sample band on the gel in reference to the point of origin (where you load your samples) or how far each DNA sample travel across agarose gel. (iv)          Explain why you would expect answer (iii) for each + sample and – sample. B.…

Answer the following questions below:

a) What are the important parameters for the eluent selection in Gel Permeation Chromotography (GPC)? Explain your answer in detail. What is an universal calibration curve and what is the advantage of using it in GPC analysis?

A plaque assay is performed beginning with 1 mL of a solution containing bacteriophages. This solution is serially diluted 3 times by combining 0.1 mL of each sequential dilution with 9.9 mL of liquid medium. Then 0.1 mL of the final dilution is plated in the plaque assay and yields 12 plaques.What is the initial density of bacteriophages in the original 1 mL? Enter your answer to two significant figures ( for example: 1.1 * 10^2)

A protein gives a single band on SDS gel electrophoresis, as shown in lanes 1 and 2 below. There is little, if any, effect from adding β-mercaptoethanol(BME) to the sample; if anything, the protein runs a little bit slower. When treated with the proteolytic enzyme thrombin and electrophoresis in the absence of BME, the protein migrates a bit more rapidly (lane 3). But if BME is present, two much more rapidly migrating bands are found (lane 4). Explain these results in terms of a model for the protein.

1) The Nde1 enzyme comes at a concentration of 40 U/ul. According to the manufacturer, the container has enough for 10,000 reactions of 2 ug of DNA, assuming that 1 U/ug of DNA is recommended. How many units in total does the packaging contain? At what concentration in U/ml does the enzyme come? If I had to use 10 U for a reaction of 10 ugs of DNA, how would I serve it? 2) A serially diluted phage solution is used to inoculate an E coli plate in the double-layer plate assay. In 24 hours you count 148 plaques. Considering that the original stock was 0.1, and that you used the sixth dilution of the series, what is the PFU for that phage in this strain of E coli? What is the dilution factor for this plate?