LaCava Research Wiki

Protocol for CALIBRATION CURVE with Native Marker

Aim: Establish a precise and reliable calibration curve

Materials: - PBS 1x pH=7.4 (previously filtered with 0,22 µm filters) - Protein sample with known masses (I used NativeMarkTM Unstained Protein standard from ThermoFisher Scientific)

Methods: - Prepare a 1:50 pre-dilution of the NM protein mixture (2 µL of NM in 98 µL of PBS) - Add 2 µL of the NM protein mix into the 18 µL buffer droplet directly into the gasket well (dilution 1:10)

Results: You should be able to obtain a calibration curve with a R2 around 0,999

Protocol for BSA measurements

Aim: Measuring BSA molecular weight with the mass photometer to see if the data are accurate and similar to the expected values.

Materials: - PBS 1x pH=7.4 (previously filtered with 0,22 µm filters) - Pierce™ Bovine Serum Albumin Standard Ampules, 2 mg/mL (Thermo ScientificTM) https://www.thermofisher.com/order/catalog/product/23209#/23209

Methods: - Prepare a pre-dilution of the BSA standard ampoule Calculations: Starting concentration 2 mg/mL = 2 g/L Final concentration = 200 nM MW = 66 kDa = 66’000 Da = 66’000 g/mol mol = g/g/mol = 2 g/L /66’000 g/mol = 0,000030 M = 30 µM = 30’000 nM Dilution Factor (DC) = 30’000 nM/200 nM = 150 Vfin = 1,5 mL = 1500 µL Vini = Vfin /DF = 1500 µL/150 = 10 10 µL BSA solution + 1490 µL PBS

- Add 2 µL of the BSA mix into the 18 µL buffer droplet directly into the gasket well (dilution 1:10 so that the final concentration is 20 nM)

Results: I expected to detect two main peaks, the first one around 66 kDa (corresponding to the monomer) and the second one around 132 kDa (corresponding to the dimer).

Protocol for Thyroglobulin measurements

Aim: Measuring Thyroglobulin molecular weight with the mass photometer to see if the data are accurate and similar to the expected values.

Materials: - PBS 1x pH=7.4 (previously filtered with 0,22 µm filters) - Thyroglobulin from bovine thyroid https://www.sigmaaldrich.com/catalog/product/sigma/t1001?lang=en&region=NL

Methods: - Prepare a stock solution of Thyroglobulin Calculations: Quantity: 0.94 mg = 0.00094 g MW = 669 kDa = 669’000 Da = 669’000 g/mol mol = g/g/mol = 0.00094/ 669’000 g/mol = 0.000 000 001 4 mol = 0.0014 µmol = 1.4 nmol Final concentration of the stock solution: 1 µM 1 µmol : 1 L = 0.0014 µmol : x x = 0.0014 L = 1.4 mL

- Prepare a pre-dilution of the stock solution Initial concentration = 1 µM = 1’000 nM Final concentration = 200 nM Dilution Factor (DC) = 1’000 nM/200 nM = 5 Vfin = 1,5 mL = 1500 µL Vini = Vfin /DF = 1500 µL/5 = 300 300 µL Thyroglobulin mix + 1200 µL PBS

- Add 2 µL of the Thyroglobulin mix into the 18 µL buffer droplet directly into the gasket well (dilution 1:10 so that the final concentration is 20 nM)

Results: I expected to detect two main peaks, the first one around 330 kDa (corresponding to the monomer) and the second one around 670 kDa (corresponding to the dimer).

Protocol for β-amylase measurements

Aim: Measuring β-amylase molecular weight with the mass photometer to see if the data are accurate and similar to the expected values.

Materials: - PBS 1x pH=7.4 (previously filtered with 0,22 µm filters) - β-amylase from sweet potato https://www.sigmaaldrich.com/catalog/product/sigma/a8781?lang=en&region=NL

Methods: - Prepare a stock solution of β-amylase Calculations: Quantity: 1.9 mg = 0.0019 g MW = 200 kDa = 200’000 Da = 200’000 g/mol mol = g/g/mol = 0.0019 g/ 200’000 g/mol = 0.000 000 009 5 mol = 0.0095 µmol = 9.5 nmol 1 µmol : 1 L = 0.0095 µmol : x x = 0.0095 L = 9.5 mL

- Prepare a pre-dilution of the stock solution Initial concentration = 1 µM = 1’000 nM Final concentration = 200 nM Dilution Factor (DC) = 1’000 nM/200 nM = 5 Vfin = 1,5 mL = 1500 µL Vini = Vfin /DF = 1500 µL/5 = 300 300 µL of β-amylase mix + 1200 µL PBS

- Add 2 µL of the β-amylase mix into the 18 µL buffer droplet directly into the gasket well (dilution 1:10 so that the final concentration is 20 nM)

Results: I expected to detect two main peaks, the first one around 121 kDa (corresponding to the dimer) and the second one around 242 kDa (corresponding to the tetramer).

Protocol for aldolase measurements

Aim: Measuring aldolase molecular weight with the mass photometer to see if the data are accurate and similar to the expected values.

Materials: - PBS 1x pH=7.4 (previously filtered with 0,22 µm filters) - Aldolase from rabbit muscle in ammonium sulfate suspension https://www.sigmaaldrich.com/catalog/product/sigma/a8811?lang=it&region=IT

Methods: - Prepare a stock solution of aldolase Calculations: Quantity: 22 mg/mL Volume of the stock = 0.61 mL Quantity of protein in the stock: 22 mg : 1 mL = x mg : 0.61 mL x mg = 13.42 mg = 0.01342 g MW = 36 kDa = 36’000 Da = 36’000 g/mol mol = g/g/mol = 0.01342 g/ 36’000 g/mol = 0.000 000 37 mol = 0.37 µmol = 370 nmol 1 µmol : 1 L = 0.37 µmol : x x = 0.37 L = 370 mL

- Prepare a pre-dilution of the stock solution Initial concentration = 1 µM = 1’000 nM Final concentration = 200 nM Dilution Factor (DC) = 1’000 nM/200 nM = 5 Vfin = 1,5 mL = 1500 µL Vini = Vfin /DF = 1500 µL/5 = 300 300 µL of aldolase mix + 1200 µL PBS

- Add 2 µL of the aldolase mix into the 18 µL buffer droplet directly into the gasket well (dilution 1:10 so that the final concentration is 20 nM)

Results: I expected to detect the main peaks corresponding to the different oligomeric states, a first one around 40 kDa (corresponding to the monomer), a second one around 80 kDa (corresponding to the dimer), a third one around 120 kDa (corresponding to the trimer), a forth one around 160 kDa (corresponding to the tetramer) and eventually a fifth one around 320 kDa (corresponding to the octamer).

Protocol for aldolase measurements at increasing [NaCl]

Aim: Measuring aldolase molecular weight with the mass photometer at increasing [NaCl] to be able to see a shift towards lower molecular weight oligomeric states.

Materials: - 0.5 M stock of HEPES (previously filtered with 0,22 µm filters) (Final concentration 20 mM) - 1 M stock solution of NaCl (Final concentrations: 0.5 and 0.25 M) - 1 µM of aldolase stock solution (Final concentration: 200 nM)

Methods: - 1 M NaCl Aldolase: C1V1=C2V2 = 1000 nM * X = 200 nM * 50 µL, x = 10 µL HEPES: C1V1=C2V2 = 0.5 M * X = 0.02 M * 50 µL, x = 2 µL NaCl = 1 M - 0.5 M NaCl Aldolase = 10 µL HEPES = 2 µL NaCl = C1V1=C2V2 = 1 M * X = 0.5 M * 50 µL, x = 25 µL Make up to volume with milliQ water = 13 µL - 0.25 M NaCl Aldolase = 10 µL HEPES = 2 µL NaCl = C1V1=C2V2 = 1 M * X = 0.25 M * 50 µL, x = 12,5 µL Make up to volume with milliQ water = 25,5 µL

- Add 2 µL of the aldolase mix at three different salt concentrations into the 18 µL buffer droplet directly into the gasket well (dilution 1:10 so that the final concentration is 20 nM)

Results: I expected to detect a progressive shift towards lower molecular weight oligomeric states, as the increase of [NaCl] progressive weaken non-covalent interactions within the molecule.