User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2013/10/23

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Objective To measure pepsin activity of 1) pepsin in solution and 2) pepsin-AuNPs in solution. We're going to try a slightly different protocol for this since the previous one see here didn't give the best results. The protocol for today is based off of this. We'll also be taking spectra of the pepsin-AuNP samples you made yesterday. Description Make a 37C water bath that can hold at least 3 test tubes (same size as you used yesterday) at a time --- START THIS FIRST Pepsin Digest Stock Solutions 2.5 w/v Hemoglobin in 0.06N HCl 1M perchloric acid 0.5mg/mL pepsin in 0.01N HCl pepsin-AuNP (at ratio that I need to decide on) Directions (You can decide how you want to split your effort here. I'd suggest doing one set, i.e. pepsin blank, pepsin trial, pepsin-AuNP blank, or pepsin-AuNP trial, at a time) Make 1mL pepsin dilutions for the regular pepsin AND the pepsin-AuNPs in 0.01N HCl 10μg/mL 15μg/mL 20μg/mL Blank (perform this for each pepsin or pepsin-AuNP dilution) Place 2.5mL hemoglobin solution in a test tube in the 37C water bath Add 5mL of perchloric acid Add 0.5mL pepsin dilution Remove from water bath after 5minutes Filter Measure the absorbance at 280 --- This will be your A280(Blank) Test (perform this for each pepsin or pepsin-AuNP dilution) Place 2.5mL hemoglobin solution in a test tube in the 37C water bath Add 0.5 mL pepsin dilution Wait 10 minutes Add 5mL of perchloric acid Remove from water bath after 5minutes Filter Measure the absorbance at 280 --- This will be your A280(Test) Calculate the number of units/mg of active enzyme in each sample Units/mg = [A280(Test) - A280(Blank)]*1000/[10minutes*mg enzyme in reaction mixture] Protein-AuNP analysis Take a picture and note physical description of each ratio Place 1mL of each ratio in an 1.5mL eppendorf tube. Centrifuge for 30 minutes (Be considerate of other uses here. Act quickly, and get as many tubes in the centrifuge as you can at once.) Take a UV-Vis spectrum of each sample (Be considerate of other users here) After taking the spectrum, save the sample in a fresh eppy tube. Synthesis of HRP-AuNPs and Hemoglobin-AuNPs Into test tube that will contain a total of 5mL add Au solution such that the final concentration is 0.25mM For Myoglobin make the following solutions to reflect an Au:Protein ratios 30 50 70 90 110 130 150 170 190 210 For HRP make the following solutions to reflect an Au:Protein ratios 230 250 270 290 310 330 350 370 390 410 Water up to 5mL Data Stock Solutions Gold 49.3 mg HAuCl4·3H2O in 50mL water ---> 2.50mM HRP 6.6 mg HRP in 100 mL water ---> 1.5μM Myoglobin 28.7mg myoglobin in 100mL water ---> 16.9μM Results We observed that Pepsin did not work as there were no nano particles formed in the tubes. The indication that no nano particles were formed is that the solutions were clean with black precipitate ins tea of a purple color. Here are the UV-Vis spectra for both: - On the graph, we can see that this is a representation of pepsin at different concentrations which means that no nano particles formed.