Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells
Abstract
:1. Introduction
2. Results and Discussion
2.1. Optimization of the BDGA Method in Order to Measure Monomer-Dimer Equilibria
2.1.1. Initial Steps in the Analysis of FFS Data
2.1.2. Two-Component BDGA of Lysate Mixtures
2.1.3. One-Component BDGA of Lysate Mixes
2.2. Improved BDGA Methodology for the Analysis of Cellular Data
2.3. Application of the Developed Methodology to the Analysis of Induced Dimerization in Cells
3. Materials and Methods
3.1. Theory of the Global Analysis of ACF and PCD
- Calculate the time dependent parameters
- Calculate single-molecular PCD for each molecular species iIn Equation (6), γ() is the incomplete gamma function and parameter Θ is varied depending on the value of the product of qeff T (from 1 to 20), see details in [38];
- Calculate PCD P(k) for each brightness component assuming the Poissonian distribution of a number of molecules in an open observation volume
- Calculate the total PCD for a molecular system. PCD of a number of independent species is given by a convolution of PCD of each species
- The correction on dead-time is performed accordingly to the following equation [39]:
3.2. Sample Preparation and Measurement
3.2.1. Plasmids and Cloning
3.2.2. Cell Culture
3.2.3. Measurement in Cell Lysate
3.2.4. Measurement of Cells
3.3. Data Analysis Procedure
3.3.1. Fitting Software
3.3.2. Analysis of the Obtained Data
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
References
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Sample | Analysis Method | Ftrip (×10−2) | τtrip (µs) | τdiff1 (µs) | N (±SD) | qtrue (×104 cpms) | SD of qtrue | χ2 |
---|---|---|---|---|---|---|---|---|
R110 | 1-component | 9.10 ± 0.78 | 6.9 ± 1.2 | 35 ± 1 | 4.50 (±0.06) | 4.04 ± 0.023 | 0.055 | 1.147 |
GFP | 1-component | 12.2 ± 0.90 | 35.1 ± 2.0 | 151 ± 4 | 3.88 (±0.09) | 3.20 ± 0.020 | 0.096 | 1.05 |
diGFP | 1-component | 7.08 ± 0.43 | 31.0 ± 2.0 | 221 ± 3 | 3.63 (±0.06) | 5.12 ± 0.023 | 0.090 | 1.233 |
50% GFP + 50% diGFP | 1-component | 8.97 ± 0.47 | 22.2 ± 2.5 | 191 ± 2 | 3.47 (±0.07) | 4.88 ± 0.025 | 0.107 | 1.193 |
2-component (r = 1.8) | 8.07 ± 0.46 | 19.1 ± 2.4 | 151 (τdiff1); 221 (τdiff2) | 2.00 (±0.18) (N1); 1.76 (±0.12) (N2) | 3.20 (q1); 5.76 (q2) | - | 1.068 |
diGFP% | τdiff (µs) | D (µm2 s−1) | qtrue (×104 cpms) | qtrue Norm. to GFP |
---|---|---|---|---|
0 | 131 ± 21 | 97.3 ± 6.5 | 4.43 ± 1.22 | 1.00 ± 0.02 |
5 | 126 ± 32 | 98.3 ± 6.6 | 5.13 ± 1.19 | 1.02 ± 0.03 |
10 | 154 ± n.a. | 99.0 ± n.a. | 5.16 ± 1.60 | 1.10 ± 0.03 |
12.5 | 132 ± 37 | 94.1 ± 3.3 | 5.19 ± 1.15 | 1.13 ± 0.03 |
33.3 | 137 ± 32 | 87.7 ± 3.9 | 6.43 ± 1.42 | 1.37 ± 0.04 |
50 | 169 ± 31 | 74.0 ± 6.0 | 5.69 ± 1.62 | 1.45 ± 0.06 |
66.7 | 168 ± 37 | 78.4 ± 0.1 | 6.20 ± 1.84 | 1.55 ± 0.25 |
87.5 | 156 ± 37 | 76.8 ± 2.2 | 8.22 ± 2.02 | 1.75 ± 0.08 |
90 | 175 ± 46 | 69.5 ± 6.9 | 8.27 ± 2.88 | 1.84 ± 0.25 |
95 | 158 ± 36 | 75.7 ± 2.4 | 8.68 ± 1.70 | 1.74 ± 0.07 |
100 | 173 ± 40 | 71.4 ± 5.9 | 8.82 ± 2.77 | 1.89 ± 0.24 |
Sample | Ftrip | τtrip (µs) | τdiff (µs) | N (range) | q (×104 cpms) | SD of q (×104 cpms) | # Traces | χ2 |
---|---|---|---|---|---|---|---|---|
GFP (trip free) | 0.241 ± 0.008 | 53.8 ± 2.8 | 381 ± 8 | 12.1 (8.5–19.9) | 4.71 ± 0.06 | 0.55 | 14 | 1.197 |
GFP (trip fixed) | 0.178 | 40.0 | 330 ± 2 | 11 (7.8–18.2) | 5.18 ± 0.07 | 0.55 | 14 | 1.108 |
diGFP | 0.128 | 61.9 | 841 ± 6 | 6.5 (4.9–7.9) | 8.32 ± 0.10 | 1.04 | 13 | 1.079 |
FKBP12-GFP + dim | 0.178 | 40.0 | 1018 ± 9 | 19 (16.5–21.7) | 6.73 ± 0.18 | 0.79 | 9 | 1.015 |
Sample | τdiff (µs) | D (µm2 s−1) | q (×104 cpms) | q Normalized to GFP | # Cells |
---|---|---|---|---|---|
GFP | 506 ± 115 | 16.7 ± 3.6 | 4.9 ± 0.6 | 1.00 ± 0.07 | 35 |
diGFP | 871 ± 336 | 10.2 ± 2.4 * | 7.8 ± 1.4 | 1.60 ± 0.25 * | 27 |
GFP + dim | 502 ± 133 | 17.1 ± 4.3 | 4.9 ± 0.6 | 1.00 ± 0.09 | 28 |
FKBP12-GFP | 882 ± 269 | 9.8 ± 2.2 | 4.8 ± 0.7 | 0.98 ± 0.09 | 31 |
FKBP12-GFP + dim | 1101 ± 336 | 7.8 ± 1.7 ** | 6.6 ± 0.9 | 1.33 ± 0.15 ** | 35 |
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Nederveen-Schippers, L.M.; Pathak, P.; Keizer-Gunnink, I.; Westphal, A.H.; van Haastert, P.J.M.; Borst, J.W.; Kortholt, A.; Skakun, V. Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells. Int. J. Mol. Sci. 2021, 22, 7300. https://doi.org/10.3390/ijms22147300
Nederveen-Schippers LM, Pathak P, Keizer-Gunnink I, Westphal AH, van Haastert PJM, Borst JW, Kortholt A, Skakun V. Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells. International Journal of Molecular Sciences. 2021; 22(14):7300. https://doi.org/10.3390/ijms22147300
Chicago/Turabian StyleNederveen-Schippers, Laura M., Pragya Pathak, Ineke Keizer-Gunnink, Adrie H. Westphal, Peter J. M. van Haastert, Jan Willem Borst, Arjan Kortholt, and Victor Skakun. 2021. "Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells" International Journal of Molecular Sciences 22, no. 14: 7300. https://doi.org/10.3390/ijms22147300