You will have to create your own blanks for expt 1B so make sure you understand

You will have to create your own blanks for expt 1b

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You will have to create your own blanks for expt 1B, so make sure you understand its purpose! (Discuss this with your TA) Does the amount of BSA present in solution have an effect on the BBG absorption spectra? Table 2.1. Reactions needed to acquire BBG absorption spectra 31
6. Tabulate the λmax, εmaxand ε595values for all of your samples. Explain the observed spectral shifts. (1B) Does SDS interfere with the absorption spectrum of BBG-BSA complex? (odd #) or Does binding of lysozyme have the same effect on BBG’s absorption spectrum as BSA? (even #)i) What effect does SDS have on the absorption spectrum of the BBG-BSA complex? (odd-numbered teams: #1, 3, 5, 7, 9, 11, 13, 15, 17) ii) What effect does protein identity (BSA vs lysozyme) have on the absorption spectrum of BBG? (even-numbered teams: # 2, 4, 6, 8, 10, 12, 14, 16, 18)
experiment 2: LIGAND BINDING 2015 7. Prepare the following 3 mL solutions according to Table 2.2 (odd)or Table 2.3 (even)The first baseline reaction has been provided – you are responsible for providing the other necessary reactions. . Sample H 2 O (mL) 2X Bradford reagent (mL) BSA 2 mg/mL (μL) SDS 1% (μL) 1 1.5 1.5 --- --- 2 1.49 1.5 --- 6 3 1.47 1.5 25 6 4 1.44 1.5 --- 60 5 1.42 1.5 25 60 BASELINE 1 3.0 2 3 4 5 Sample H 2 O (mL) 2X Bradford reagent (mL) BSA 2 mg/mL (μL) Lysozyme 10 mg/mL (μL) 1 1.5 1.5 --- --- 2 1.47 1.5 25 --- 3 1.44 1.5 125 --- 4 1.42 1.5 --- 25 BASELINE 1 3.0 2 3 4 Table 2.2. Reactions needed to acquire BBG absorption spectra for experiment 1B (i) – odd # teams Table 2.3. Reactions needed to acquire BBG absorption spectra for experiment 1B (ii) – even # teams 33
experiment 2: LIGAND BINDING 2015 8. When adding the dye, mix thoroughlyusing a stirring magnet (this is very important for good results!). 9. Record the spectrum of each using the corresponding baseline samples in the reference cuvette (Table 2.2 or Table 2.3). Save these spectra in memory in case you need to return to them later during your experiments. 10. To determine the effect of your experimental treatment on the BBG absorption spectra, superimpose the corrected spectra (after the baseline has been subtracted) of the appropriate samples (it will be up to you to determine which curve to superimpose). 11. Tabulate the λmax, εmaxand ε595values for all of your samples. Explain the observed spectral shifts.

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