Reagent Setup

II.3.1. General Reagent

Laemmli buffer (4x, for protein sample loading in SDS-PAGE) contains 8% SDS, 40%

glycerol, 20% β-mercaptoethanol, 0.008% Bromophenol Blue, 0.25 M Tris, pH 6.8.

II.3.2. Reagents for Protein Expressions

Carbenicillin solution, 1000x stock: dissolve the carbenicillin powder at 50 mg ml⁻¹ in deionized water; aliquot in 1-ml portions. This solution can be stored at -20°C indefinitely. Caution: it should be freshly diluted 1000x into the medium or the agar right before use.

Chloramphenicol solution, 1000x stock: dissolve the chloramphenicol powder at 35

mg ml⁻¹ in pure ethanol; aliquot in 1-ml portions. This solution can be stored at -20 °C indefinitely. Caution: it should be freshly diluted 1000x into the medium right before use.

LB broth: dissolve 25 g Miller’s LB broth powder with 1 liter ddH2O in a 2000-ml cell culture flask, autoclaved at 121°C, 20 min, then cooled at room temperature. It could be kept at 4°C for 2-3 days.

17

II.3.3. Reagents for Affinity Chromatography

Amylose resin column buffer contains 20 mM Tris, 200 mM NaCl, and 1 mM EDTA,

pH 7.4.

Factor Xa reaction buffer contains 100 mM NaCl, 50 mM Tris, and 5 mM CaCl2; the pH is adjusted with 6 M HCl to 8.0.

Note: for each dialysis using the SnakeSkin tube, the exchanged buffer is recommended to have 5 liters or more, for the overnight dialysis at 4°C.

PMSF solution, 500x stock: dissolved in DMSO to make 500 mM; 1000-μl aliquots

can be stored at -20°C for months.

II.3.4. Reagent for Gel-Filtration

FPLC working buffer contains 50 mM Tris/100 mM NaCl, pH 8.0. Note: prepare 500

ml the working buffer and filter it by the 0.22-μm, 150-ml vacuum bottle top filter before use in machine; it could be kept at 4°C for weeks.

18

II.3.5. Reagents for MS Preparation

The following solutions are used in sample desalting with the ZipTip. All solutions are freshly prepared before use.

1% TFA (10x): to acidify the sample for binding onto ZipTip. It could be prepared by

mixing 990 μl ddH2O and 10 μl 100% TFA.

Wetting solution: 100% acetonitrile (ACN), distributed in the 4-ml glass sample vials.

Equilibration solution: 0.1% TFA in ddH2O, could be prepared by mixing 180 μl ddH2O and 20 μl 1% TFA (see above for its preparation).

Wash solution: 0.1% TFA in ddH2O, could be prepared by mixing 450 μl ddH2O and 50 μl 1% TFA (see above for its preparation).

Elution solution: 1% formic acid (FA)/50% methanol (MeOH), it could be prepared by

mixing 49 μl ddH2O, 50 μl MeOH, and 1 μl FA.

19

II.3.6. Reagents for Circular Dichroism

Phosphate buffer 5 L (50 mM NaH2PO4/Na2HPO4, 10 mM NaCl, pH 7.1): dissolve

15 g NaH2PO4, 17.7 g Na2HPO4 and 2.9 g NaCl in ~4.9 L ddH2O with stir bar, and adjust the pH with 5 M NaOH using a dropper. Caution: fine tune the pH with a pipette when it is near 7, and add ddH2O to a final volume of 5 L.

LC3 protein solution in the phosphate buffer or in the denaturant (the urea or the

GuHCl): to prepare the protein solution in the phosphate buffer, simply dialyze it using dialysis tube; to prepare the LC3 solution in the 8 M urea/50 mM phosphate/10 mM NaCl or in the 6 M GuHCl/50 mM phosphate/10 mM NaCl, dialyze the LC3 solution with the denaturant using the SnakeSkin tube 7K MWCO. Note: to prepare the protein solution in the 8 M urea or in the 6 M GuHCl, dialysis using the centrifugation filter device could be reliable and more efficient. Caution: do not dialyze the protein in the urea (or the GuHCl) solution with the denaturant-free buffer by centrifugation, since this might cause massive protein aggregation on the filter membrane, leading to significant loss.

20

8 M urea in 50 mM sodium phosphate/10 mM NaCl buffer, pH 7.1, 45 ml: note

since the urea powder needed to make a 8 M solution takes a significant volume in the final solution (e.g., 21.6 g urea powder to make 45 ml 8 M urea), the phosphate/NaCl buffer to be included in the urea solution is prepared as the 10x stock (500 mM sodium phosphate/100 mM NaCl) in advanced. For a final volume of 45 ml, 21.6 g urea powder could first be measured in a 50-ml tube, followed by adding 4.5 ml the 10x phosphate/NaCl buffer and some distilled water to volume ~44 ml. After pH adjustment to 7.1 with 5 M NaOH, replenish the solution volume to 45 ml with distilled water. Note:

urea dissolving in water is an exothermic reaction, which could be promoted by shaking at room temperature. Caution: urea is not very stable in water, and is thus recommended freshly made before use. An alternative is to prepare a 100-ml stock, distributing it into 5-6 aliquots, and it can be stored in -20°C for a month.

6 M GuHCl in 50 mM sodium phosphate/10 mM NaCl buffer, pH 7.1, 45 ml: note

since the GuHCl powder needed to make a 6 M solution takes a significant volume in the final solution (e.g., 25.8 g GuHCl powder to make 45 ml 6 M GuHCl), the phosphate/NaCl buffer to be included in the GuHCl solution is prepared as the 10x stock (500 mM sodium phosphate/100 mM NaCl) in advanced. For a final volume of 45 ml, 25.8 g GuHCl powder could first be measured in a 50-ml tube, followed by adding

21

4.5 ml the 10x phosphate/NaCl buffer and some distilled water to volume ~44 ml. After pH adjustment to 7.1 with 5 M NaOH, replenish the solution volume to 45 ml with distilled water. 6 M GuHCl can be stored at 4°C for weeks. Caution: the purity of GuHCl powder used should exceed 99.9%, since the impurities in the lower grade GuHCl might have high absorbance in the CD that could generate large noise in signals.

II.3.7. Reagents for PKA Reaction

ATP solution: 0.25 mM ATP is prepared by diluting 0.1 M ATP with 25 mM Mg(OAc)2, pH 7.0. Note: ATP could be stored at -20°C for months.

Kemptide: prepare 300 μM stock by dissolving the purchased Kemptide 1 mg with the

PKA reaction buffer 4.318 ml, stored at -20 °C in 500 μl aliquots. Note: snap freezing the peptide with liquid nitrogen is recommended before storage.

PKA reaction buffer (5x): 40 mM MOPS, 1 mM EDTA, pH 7.0.

PKA stock: 10x serially dilute the purchased PKA (0.432 μg/μl) with the buffer

containing 20 mM MOPS, pH 7.0, 1 mM EDTA, 0.01% Brij-35, 5% glycerol, 0.1%

β-mercaptoethanol, and 1 mg/ml BSA, to obtain 4.32 ng/μl PKA aliquots, which could be stored at -20°C for months. Caution: this buffer should prevent light exposure and snap freezing the enzyme with liquid nitrogen is recommended before storage.

22