Drybench Assay.
Apparent Tm from a thermal shift melt
Two apparent Tms from a fluorescence melt in a window you choose, the sigmoid midpoint and the derivative extremum, with ΔTm against a reference condition.
Two apparent Tms from a fluorescence melt you measured, in a temperature window you type for each condition: the midpoint of a fitted sigmoid and the temperature where the signal changes fastest, side by side, with ΔTm against a reference condition: apparent values under your own assay's conditions.
Thermal shift: apparent Tm
Paste a fluorescence melt for one or more conditions. Each condition's curve is analysed in a temperature window you type, and two apparent Tms are reported side by side: the midpoint of a fitted sigmoid and the temperature where the signal changes fastest.
Melt readings
Columns: temperature (°C), fluorescence, and optionally condition. One reading per row, temperatures increasing within each condition. Each condition is one curve, fitted on its own; enter replicate wells as separate conditions. CSV or tab-separated text; at most 24 conditions and 5,000 rows.
Synthetic demonstration data: two melts generated from known sigmoids (apparent Tm 52.0 and 56.3 °C) with a fixed noise vector; not experimental evidence.
Window, direction and conditions
Declare how your signal changes as the protein unfolds. The derivative extremum follows this declaration; a fitted change in the other direction is refused.
Window for each condition
Type the lowest and highest temperature to analyse for each condition, both included. Keep both plateaus of one transition inside the window and leave out any decline after it.
Optional. Give the conditions that are replicate wells of one sample the same group name; their sigmoid Tms are summarised as their mean and SD with n.
ΔTm is each condition's apparent Tm minus the reference's, by the same method, when both have one.
Assay conditions
Recorded with the result as you type them; a condition left empty is reported as not stated.
Melt readings
Enter a reason, then choose a reading. The raw value is retained; changing a decision clears the current result.
| Source row | Temperature (°C) | Fluorescence | Fitted | Residual | Decision |
|---|---|---|---|---|---|
| Protein alone, 141 readings | |||||
| 2 | 25 | 1039.5440344422368 | – | – | |
| 3 | 25.5 | 1029.8142382484868 | – | – | |
| 4 | 26 | 1011.8721254443751 | – | – | |
| 5 | 26.5 | 983.2546449461112 | – | – | |
| 6 | 27 | 960.2120408416275 | – | – | |
| 7 | 27.5 | 949.724004587505 | – | – | |
| 8 | 28 | 1127.5630946759914 | – | – | |
| 9 | 28.5 | 1002.8590136941102 | – | – | |
| 10 | 29 | 974.3534009310376 | – | – | |
| 11 | 29.5 | 923.3715648457811 | – | – | |
| 12 | 30 | 1051.7884645063243 | – | – | |
| 13 | 30.5 | 972.649971207578 | – | – | |
| 14 | 31 | 1005.348671491945 | – | – | |
| 15 | 31.5 | 957.7729063430054 | – | – | |
| 16 | 32 | 1022.7313742648446 | – | – | |
| 17 | 32.5 | 1008.6804990007969 | – | – | |
| 18 | 33 | 1018.9426723369627 | – | – | |
| 19 | 33.5 | 1097.4291332265739 | – | – | |
| 20 | 34 | 860.1249065412027 | – | – | |
| 21 | 34.5 | 1020.7504529934123 | – | – | |
| 22 | 35 | 985.375609360604 | – | – | |
| 23 | 35.5 | 975.8108138457081 | – | – | |
| 24 | 36 | 969.142442519976 | – | – | |
| 25 | 36.5 | 984.862825425235 | – | – | |
| 26 | 37 | 950.4906420199469 | – | – | |
| 27 | 37.5 | 1035.7880349465893 | – | – | |
| 28 | 38 | 1048.9407957490998 | – | – | |
| 29 | 38.5 | 999.2109416703196 | – | – | |
| 30 | 39 | 1055.8430648321234 | – | – | |
| 31 | 39.5 | 1002.2637929363033 | – | – | |
| 32 | 40 | 1016.07243367395 | – | – | |
| 33 | 40.5 | 983.2457956226344 | – | – | |
| 34 | 41 | 1048.9104898226954 | – | – | |
| 35 | 41.5 | 972.7533934161493 | – | – | |
| 36 | 42 | 963.7287286167502 | – | – | |
| 37 | 42.5 | 995.5386863579554 | – | – | |
| 38 | 43 | 1113.1270304425743 | – | – | |
| 39 | 43.5 | 1074.6525528348313 | – | – | |
| 40 | 44 | 1139.0427966806412 | – | – | |
| 41 | 44.5 | 1126.9920944540459 | – | – | |
| 42 | 45 | 1203.8282937041422 | – | – | |
| 43 | 45.5 | 1209.1453184549632 | – | – | |
| 44 | 46 | 1230.0385171170913 | – | – | |
| 45 | 46.5 | 1424.4252846245465 | – | – | |
| 46 | 47 | 1472.7340833480864 | – | – | |
| 47 | 47.5 | 1633.373456786308 | – | – | |
| 48 | 48 | 1855.8461661508773 | – | – | |
| 49 | 48.5 | 2064.8281254531794 | – | – | |
| 50 | 49 | 2314.6428599199285 | – | – | |
| 51 | 49.5 | 2620.8988307872364 | – | – | |
| 52 | 50 | 2981.3020297246744 | – | – | |
| 53 | 50.5 | 3424.067676388837 | – | – | |
| 54 | 51 | 3956.8327143720107 | – | – | |
| 55 | 51.5 | 4435.438369088637 | – | – | |
| 56 | 52 | 5047.867624760639 | – | – | |
| 57 | 52.5 | 5554.865234083192 | – | – | |
| 58 | 53 | 6108.628626866059 | – | – | |
| 59 | 53.5 | 6638.496841689644 | – | – | |
| 60 | 54 | 7084.059417436019 | – | – | |
| 61 | 54.5 | 7454.2707470766 | – | – | |
| 62 | 55 | 7767.535169226428 | – | – | |
| 63 | 55.5 | 7937.206314697115 | – | – | |
| 64 | 56 | 8224.355191279905 | – | – | |
| 65 | 56.5 | 8345.014644431027 | – | – | |
| 66 | 57 | 8536.70389649651 | – | – | |
| 67 | 57.5 | 8588.75307963446 | – | – | |
| 68 | 58 | 8717.700498834785 | – | – | |
| 69 | 58.5 | 8826.828624418295 | – | – | |
| 70 | 59 | 8841.627952899917 | – | – | |
| 71 | 59.5 | 8911.424972237173 | – | – | |
| 72 | 60 | 8872.273319790049 | – | – | |
| 73 | 60.5 | 8965.000557900028 | – | – | |
| 74 | 61 | 8946.517570258318 | – | – | |
| 75 | 61.5 | 8930.631803484954 | – | – | |
| 76 | 62 | 8962.742349493074 | – | – | |
| 77 | 62.5 | 8909.920984554643 | – | – | |
| 78 | 63 | 9010.821887351605 | – | – | |
| 79 | 63.5 | 9043.800988268726 | – | – | |
| 80 | 64 | 8949.746757637758 | – | – | |
| 81 | 64.5 | 9029.838602310818 | – | – | |
| 82 | 65 | 8985.295031039846 | – | – | |
| 83 | 65.5 | 8944.76649253206 | – | – | |
| 84 | 66 | 8995.44394698314 | – | – | |
| 85 | 66.5 | 8943.828184924283 | – | – | |
| 86 | 67 | 9028.834009233626 | – | – | |
| 87 | 67.5 | 9100.411457487104 | – | – | |
| 88 | 68 | 9051.091836607578 | – | – | |
| 89 | 68.5 | 8966.977184809442 | – | – | |
| 90 | 69 | 8965.298624036337 | – | – | |
| 91 | 69.5 | 8987.773919778938 | – | – | |
| 92 | 70 | 9008.576076227431 | – | – | |
| 93 | 70.5 | 8985.288091829112 | – | – | |
| 94 | 71 | 9048.596183776648 | – | – | |
| 95 | 71.5 | 9012.293566818145 | – | – | |
| 96 | 72 | 9016.159278620411 | – | – | |
| 97 | 72.5 | 8964.941556925962 | – | – | |
| 98 | 73 | 9053.0243225835 | – | – | |
| 99 | 73.5 | 9035.689350473649 | – | – | |
| 100 | 74 | 8976.772778089784 | – | – | |
| 101 | 74.5 | 9001.089604940122 | – | – | |
| 102 | 75 | 9053.80933680524 | – | – | |
| 103 | 75.5 | 8992.253945639573 | – | – | |
| 104 | 76 | 8949.755080666915 | – | – | |
| 105 | 76.5 | 9057.430972122087 | – | – | |
| 106 | 77 | 9064.724669935838 | – | – | |
| 107 | 77.5 | 8944.559672330179 | – | – | |
| 108 | 78 | 8916.509982360281 | – | – | |
| 109 | 78.5 | 8974.079030932317 | – | – | |
| 110 | 79 | 9057.756326454479 | – | – | |
| 111 | 79.5 | 9024.87467057753 | – | – | |
| 112 | 80 | 9012.881031878484 | – | – | |
| 113 | 80.5 | 8993.7733356884 | – | – | |
| 114 | 81 | 9009.50702546964 | – | – | |
| 115 | 81.5 | 8967.707839785788 | – | – | |
| 116 | 82 | 8960.846987249111 | – | – | |
| 117 | 82.5 | 8999.840367092464 | – | – | |
| 118 | 83 | 9023.944218805289 | – | – | |
| 119 | 83.5 | 8983.560725636804 | – | – | |
| 120 | 84 | 9011.83436025096 | – | – | |
| 121 | 84.5 | 9094.933990338572 | – | – | |
| 122 | 85 | 9005.781015006585 | – | – | |
| 123 | 85.5 | 8969.566027127814 | – | – | |
| 124 | 86 | 9019.535084122888 | – | – | |
| 125 | 86.5 | 8997.325009504366 | – | – | |
| 126 | 87 | 9033.686935957236 | – | – | |
| 127 | 87.5 | 8970.694221841048 | – | – | |
| 128 | 88 | 8984.80154807132 | – | – | |
| 129 | 88.5 | 8974.609876174174 | – | – | |
| 130 | 89 | 9000.228080469165 | – | – | |
| 131 | 89.5 | 9036.312909697506 | – | – | |
| 132 | 90 | 8982.584235608014 | – | – | |
| 133 | 90.5 | 8947.356353293222 | – | – | |
| 134 | 91 | 8945.572516961107 | – | – | |
| 135 | 91.5 | 8966.66618179219 | – | – | |
| 136 | 92 | 9000.895279992905 | – | – | |
| 137 | 92.5 | 9000.333028500463 | – | – | |
| 138 | 93 | 8989.26173169281 | – | – | |
| 139 | 93.5 | 9003.139506569398 | – | – | |
| 140 | 94 | 8956.560016391493 | – | – | |
| 141 | 94.5 | 9047.187448922537 | – | – | |
| 142 | 95 | 9012.348843345819 | – | – | |
| Protein + ligand, 141 readings | |||||
| 143 | 25 | 1039.5418117115703 | – | – | |
| 144 | 25.5 | 1029.8113038158037 | – | – | |
| 145 | 26 | 1011.8682514280558 | – | – | |
| 146 | 26.5 | 983.2495304986574 | – | – | |
| 147 | 27 | 960.2052887866435 | – | – | |
| 148 | 27.5 | 949.7150905761035 | – | – | |
| 149 | 28 | 1127.5513264674698 | – | – | |
| 150 | 28.5 | 1002.8434773988834 | – | – | |
| 151 | 29 | 974.3328900414506 | – | – | |
| 152 | 29.5 | 923.3444865439834 | – | – | |
| 153 | 30 | 1051.7527159741376 | – | – | |
| 154 | 30.5 | 972.6027763343545 | – | – | |
| 155 | 31 | 1005.2863653021294 | – | – | |
| 156 | 31.5 | 957.690650407096 | – | – | |
| 157 | 32 | 1022.6227810011796 | – | – | |
| 158 | 32.5 | 1008.5371357062055 | – | – | |
| 159 | 33 | 1018.7534064546916 | – | – | |
| 160 | 33.5 | 1097.1792680488184 | – | – | |
| 161 | 34 | 859.7950403006913 | – | – | |
| 162 | 34.5 | 1020.3149728724196 | – | – | |
| 163 | 35 | 984.8007036899855 | – | – | |
| 164 | 35.5 | 975.051848517067 | – | – | |
| 165 | 36 | 968.1404985509565 | – | – | |
| 166 | 36.5 | 983.5401299814307 | – | – | |
| 167 | 37 | 948.744540147553 | – | – | |
| 168 | 37.5 | 1033.4830374816559 | – | – | |
| 169 | 38 | 1045.8980921457921 | – | – | |
| 170 | 38.5 | 995.1945742812535 | – | – | |
| 171 | 39 | 1050.5417100183279 | – | – | |
| 172 | 39.5 | 995.2667673248918 | – | – | |
| 173 | 40 | 1006.8381199368018 | – | – | |
| 174 | 40.5 | 971.0601360478394 | – | – | |
| 175 | 41 | 1032.8324985088732 | – | – | |
| 176 | 41.5 | 951.5437577767898 | – | – | |
| 177 | 42 | 935.7564788546235 | – | – | |
| 178 | 42.5 | 958.6595898164563 | – | – | |
| 179 | 43 | 1064.525827372086 | – | – | |
| 180 | 43.5 | 1010.6394630991128 | – | – | |
| 181 | 44 | 1054.7930812166649 | – | – | |
| 182 | 44.5 | 1016.2162456621442 | – | – | |
| 183 | 45 | 1058.36039883674 | – | – | |
| 184 | 45.5 | 1018.439587304337 | – | – | |
| 185 | 46 | 980.5710067830976 | – | – | |
| 186 | 46.5 | 1099.0126919374682 | – | – | |
| 187 | 47 | 1049.8084844630703 | – | – | |
| 188 | 47.5 | 1086.2960992979088 | – | – | |
| 189 | 48 | 1152.4087328870382 | – | – | |
| 190 | 48.5 | 1167.1747490987693 | – | – | |
| 191 | 49 | 1179.9362550655362 | – | – | |
| 192 | 49.5 | 1203.1129951057299 | – | – | |
| 193 | 50 | 1234.489466622952 | – | – | |
| 194 | 50.5 | 1307.2457251149724 | – | – | |
| 195 | 51 | 1440.2191116383628 | – | – | |
| 196 | 51.5 | 1507.2023826634745 | – | – | |
| 197 | 52 | 1720.0588872911367 | – | – | |
| 198 | 52.5 | 1867.0270642850235 | – | – | |
| 199 | 53 | 2127.97340851866 | – | – | |
| 200 | 53.5 | 2456.3051393950714 | – | – | |
| 201 | 54 | 2808.7996856247155 | – | – | |
| 202 | 54.5 | 3202.483707266423 | – | – | |
| 203 | 55 | 3653.718586495894 | – | – | |
| 204 | 55.5 | 4063.9491765486105 | – | – | |
| 205 | 56 | 4673.972124351904 | – | – | |
| 206 | 56.5 | 5173.8739650370835 | – | – | |
| 207 | 57 | 5773.120666103593 | – | – | |
| 208 | 57.5 | 6234.62986479282 | – | – | |
| 209 | 58 | 6753.234381321419 | – | – | |
| 210 | 58.5 | 7216.950403279472 | – | – | |
| 211 | 59 | 7542.6840547518705 | – | – | |
| 212 | 59.5 | 7876.9401995531525 | – | – | |
| 213 | 60 | 8057.194044918658 | – | – | |
| 214 | 60.5 | 8328.338810313651 | – | – | |
| 215 | 61 | 8452.64643762386 | – | – | |
| 216 | 61.5 | 8549.610025168397 | – | – | |
| 217 | 62 | 8670.034089796492 | – | – | |
| 218 | 62.5 | 8685.79921895815 | – | – | |
| 219 | 63 | 8839.652914911558 | – | – | |
| 220 | 63.5 | 8913.329739705854 | – | – | |
| 221 | 64 | 8850.445938486007 | – | – | |
| 222 | 64.5 | 8954.347965730014 | – | – | |
| 223 | 65 | 8927.955524788298 | – | – | |
| 224 | 65.5 | 8901.24276688759 | – | – | |
| 225 | 66 | 8962.42382495609 | – | – | |
| 226 | 66.5 | 8918.786441554777 | – | – | |
| 227 | 67 | 9009.848427984623 | – | – | |
| 228 | 67.5 | 9086.02058161931 | – | – | |
| 229 | 68 | 9040.185528889811 | – | – | |
| 230 | 68.5 | 8958.712751748615 | – | – | |
| 231 | 69 | 8959.03671802703 | – | – | |
| 232 | 69.5 | 8983.029661619956 | – | – | |
| 233 | 70 | 9004.98184466774 | – | – | |
| 234 | 70.5 | 8982.565230103522 | – | – | |
| 235 | 71 | 9046.533506294127 | – | – | |
| 236 | 71.5 | 9010.73104310158 | – | – | |
| 237 | 72 | 9014.975653995869 | – | – | |
| 238 | 72.5 | 8964.044963874956 | – | – | |
| 239 | 73 | 9052.345162422796 | – | – | |
| 240 | 73.5 | 9035.174897602938 | – | – | |
| 241 | 74 | 8976.383090674473 | – | – | |
| 242 | 74.5 | 9000.794426120343 | – | – | |
| 243 | 75 | 9053.585746737557 | – | – | |
| 244 | 75.5 | 8992.08458258031 | – | – | |
| 245 | 76 | 8949.626793263731 | – | – | |
| 246 | 76.5 | 9057.333798433487 | – | – | |
| 247 | 77 | 9064.651063994683 | – | – | |
| 248 | 77.5 | 8944.503918246965 | – | – | |
| 249 | 78 | 8916.467750500677 | – | – | |
| 250 | 78.5 | 8974.047041722126 | – | – | |
| 251 | 79 | 9057.732095715031 | – | – | |
| 252 | 79.5 | 9024.856316621157 | – | – | |
| 253 | 80 | 9012.867129387263 | – | – | |
| 254 | 80.5 | 8993.762805030767 | – | – | |
| 255 | 81 | 9009.499048860644 | – | – | |
| 256 | 81.5 | 8967.701797780872 | – | – | |
| 257 | 82 | 8960.842410640074 | – | – | |
| 258 | 82.5 | 8999.836900470134 | – | – | |
| 259 | 83 | 9023.941592959429 | – | – | |
| 260 | 83.5 | 8983.558736649944 | – | – | |
| 261 | 84 | 9011.832853662661 | – | – | |
| 262 | 84.5 | 9094.932849150411 | – | – | |
| 263 | 85 | 9005.780150596313 | – | – | |
| 264 | 85.5 | 8969.565372367157 | – | – | |
| 265 | 86 | 9019.53458816451 | – | – | |
| 266 | 86.5 | 8997.324633833181 | – | – | |
| 267 | 87 | 9033.68665139941 | – | – | |
| 268 | 87.5 | 8970.694006298416 | – | – | |
| 269 | 88 | 8984.801384805292 | – | – | |
| 270 | 88.5 | 8974.609752505852 | – | – | |
| 271 | 89 | 9000.227986794724 | – | – | |
| 272 | 89.5 | 9036.312838742382 | – | – | |
| 273 | 90 | 8982.584181861981 | – | – | |
| 274 | 90.5 | 8947.356312582477 | – | – | |
| 275 | 91 | 8945.572486124138 | – | – | |
| 276 | 91.5 | 8966.66615843426 | – | – | |
| 277 | 92 | 9000.895262300088 | – | – | |
| 278 | 92.5 | 9000.333015098771 | – | – | |
| 279 | 93 | 8989.261721541496 | – | – | |
| 280 | 93.5 | 9003.13949888013 | – | – | |
| 281 | 94 | 8956.56001056714 | – | – | |
| 282 | 94.5 | 9047.187444510795 | – | – | |
| 283 | 95 | 9012.348840004077 | – | – | |
What this analysis does not do
- It does not fit two transitions or a plate of wells in one curve; each condition is one curve.
- It does not report ΔG, ΔH, a binding constant or an affinity, and does not say that a condition stabilises the protein.
- It does not smooth the readings or fit a sloping baseline; leave a decline after the transition out of the window.
- It does not pool replicate wells' readings into one curve; it summarises them only as the mean and SD of their sigmoid Tms. Enter each well as its own condition.
- It does not report an interval from a single well: adjacent readings of a melt can be correlated, which makes such an interval too narrow, and no check on the residuals reliably detects this.
Method
Two apparent Tms are found for each condition's melt inside the window typed for it. The sigmoid midpoint comes from F(T) = F_lo + (F_hi − F_lo)/(1 + exp((Tm − T)/k)) fitted by ordinary least squares with a dimensionless bounded Levenberg–Marquardt search (k searched on the log scale, three starts, verified convergence required). The derivative extremum is the temperature at which the quadratic-interpolant derivative of the signal, signed by the declared direction, is largest; its sampling resolution is half the larger adjacent temperature step; this is not an uncertainty interval. Readings are not smoothed. A curve is refused, with no Tm, below 10 readings in the window, with repeated temperatures, without a clear transition, with a second transition, with a change opposite to the declared direction, with the transition at the window's edge, or without both plateaus. A difference between the two values larger than 1 °C or two temperature steps is flagged and both are kept. Checked against SciPy on 13 synthetic melts.
1.1.0
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Worked example
- The synthetic melts loaded below (one condition generated with an apparent Tm of 52.0 °C, one at 56.3 °C, with a fixed noise vector) give a sigmoid midpoint of 51.97 °C and a derivative extremum of 52 °C for the first, and ΔTm of 4.33 °C (sigmoid midpoint) and 4 °C (derivative extremum) for the second. Computed when this page is built by the same engine the app uses, and checked against SciPy on the published fixture.
- A flat curve, two transitions, a window that cuts off a plateau, repeated temperatures and a change against the declared direction are refused with no number; a decline after the transition is flagged when the two values part: the fixture holds each case.
Method
- Apparent Tm of a fluorescence melt (sigmoid midpoint and derivative extremum). Niesen FH, Berglund H, Vedadi M (2007), Nature Protocols 2:2212–2221Checked against SciPy curve_fit (trf, bounded) and hand central differences on 13 synthetic melts: Tm within 1e-3 °C, ln k within 1e-4, the derivative extremum exact, refusals and the disagreement flag exact; and against R 4.4.3 nls(SSfpl) on the 7 fitted melts: Tm within 1e-3 °C, ln k within 1e-4, plateaus within 1e-4 of the span (12 October 2026).
Limits
- For research and education only, and not medical advice: a C50 estimated here describes this assay under these conditions and is not a binding constant.