Name: regeneration of arrayed gold microelectrodes equipped for a real-time cell analyzer
Uploaded: 2018-03-12T12:30:00
Description: Watch this Scientific Journal Video about Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer at JoVE.com

Arbejdet var støttet af National Natural Science Foundation of China (U1703118), et projekt finansieret af prioriterede akademiske Program udvikling af Jiangsu videregående institutioner (PAPD), Jiangsu Shuangchuang Program, åbne midler fra staten nøgle Laboratorium for kemo/Biosensing og kemometri (2016015) og det nationale laboratorium af Biomacromolecules (2017kf05) og Jiangsu Specially-Appointed Professor projekt, Kina.

Bemærk: generelt regenereringsprocessen omfatter trypsin fordøjelse og føres skridt med ethanol og vand. Fordøjelsestid ændres afhængig af antallet af celler, der bruges, og typen og antallet af celler, der bruges kan variere afhængigt af de eksperimentelle formål. Det anbefales at tjekke den regenererede mikrochips ved hjælp af optiske og elektrokemiske metoder til at optimere regenerering betingelser. Under eksperimentet, opløselige og uopløselige kemikalier kan være involveret, og her disse to typiske tilf...

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Vi opsummerede flere tilgængelige metoder17,23,24,25,26 for regenererende mikrochips i tabel 1. Dybest set, disse metoder involveret relativt barske eksperimentelle betingelser til at opnå en fuldstændig regenerering af chips på grund af tilstedeværelsen af stærke molekyle-molekyle interaktioner som immun komplekset brugt til SPR chips. ...

På grund af dens effektive og mindre arbejdskrævende eksperimenterende proces, har etiket-gratis celle-baseret teknologi oplevet hurtige vækst i det sidste årti for såvel analytisk som screening formål, såsom i aspekt af proteomics1,2 , drug delivery3, etc.4,5 sammenlignet med traditionelle biokemiske metoder med henblik på celle analyse, etiket-free real-time celle assay med prototypen udviklet af Giaever og kolleger tidligere6 er baseret på princippet om registrering af elektrisk signal ændringer på overfladen af cellen-attached mikrochips, som tillader en kontinuerlig måling af cellevækst eller migration i en kvantitativ måde. Efter denne strategi, en real-time celle elektroniske sensing (RT-CES) system ved hjælp af elektrisk impedans-baseret detektion princippet blev indført7,8 og mere for nylig et kommercielt real-time celle analyzer (RTCA) blev lanceret for laboratoriet forskning9.
Kommercielle real-time celle analyzer primært læser celler evoked signaler af elektrisk impedans, der skyldes de fysiologiske ændringer af rugede celler herunder celleproliferation, migration, levedygtighed, morfologi og fastholdelse på den overfladen af mikrochips10,11. Elektriske signaler konverteres yderligere af analyzer til en dimensionsløs parameter med navnet celle indeks (CI) til at vurdere celle status. Ændringen af impedans af mikrochips afspejler hovedsagelig den lokale ionisk miljø overdækket celler i grænsefladen elektrode/løsning. Derfor, den analytiske ydeevne af celle analyzer er stærkt afhængig af core sensing enhed, den engangs mikrochips (dvs., såkaldt elektronisk plader, fx, 96/16/8-godt), som er lavet af klædt guld microelectrodes lithographically udskrives i bunden af inkubation brønde. De guld microelectrodes samles i en cirkel-on-line format (figur 1) og dækker det meste af arealet af inkubation wells, som giver mulighed for dynamisk og følsomme påvisning af tilknyttede celler3,12,13 ,14. CI vil øge for flere overfladedækning af celler på chippen og falde når cellerne er udsat for en giftstoffet resulterer i apoptose. Selvom real-time celle analyzer har ofte været brugt til at bestemme cytotoksicitet11 og neurotoksicitet15 og giver flere kinetic oplysninger end klassisk slutpunkter metode, er engangs elektronisk plader de dyreste forbrugsvarer.
Indtil nu har der været nogen tilgængelige metoder til regenerering af den elektroniske plade, som er sandsynligvis skyldes, at barske regenerering betingelser, såsom piranha løsning eller eddikesyre er involveret16,17, 18, som kan ændre den elektriske status af guld mikrochips. En mild og effektiv metode til at fjerne de vedhængende celler og andre stoffer fra overfladen af guld chips vil derfor være ønskeligt for elektroniske plade regenereringsprocessen. Vi har for nylig udviklet en protokol med henblik på regenerering af engangstændere elektronisk plader ved hjælp af ikke-korroderende reagenser og regenereret chips var karakteriseret ved elektrokemiske samt optisk metoder19. Ved hjælp af let tilgængelige og moderat laboratoriereagenser herunder trypsin og ethanol, har vi etableret en generel metode for at regenerere den kommercielle elektroniske plade uden bivirkninger, som er anvendt med succes til at regenerere de to hovedtyper af elektroniske plade (både 16 og L8) bruges til RTCA.

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			<td style="width:317px;">Bio-Rad</td>
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			<td style="width:192px;">Panning: 1:1,000; Staining: 1:500</td>
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			<td style="width:317px;">Abcam</td>
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			<td style="width:317px;">Abcam&#160;</td>
			<td style="width:263px;">Cat# AB15580</td>
			<td style="width:192px;">Staining: 1:500</td>
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			<td height="21" style="height:21px;width:293px;">Alexa Fluor Donkey anti-mouse 594</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# 11055</td>
			<td style="width:192px;">Staining: 1:500</td>
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			<td height="21" style="height:21px;width:293px;">Alexa Fluor Donkey anti-goat 488</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# A-21203</td>
			<td style="width:192px;">Staining: 1:500</td>
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			<td height="21" style="height:21px;width:293px;">Alexa Fluor Donkey anti-Rabbit 647</td>
			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# A-31573</td>
			<td style="width:192px;">Staining: 1:500</td>
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			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# P6838</td>
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			<td style="width:263px;">Cat# 20606</td>
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			<td style="width:317px;">Invitrogen</td>
			<td style="width:263px;">Cat# C3100MP</td>
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			<td height="21" style="height:21px;width:293px;">Ethidium homodimer-1</td>
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			<td height="21" style="height:21px;width:293px;">Percoll PLUS</td>
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			<td style="width:263px;">Cat# 17-5445-02</td>
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			<td style="width:263px;">Cat# 15140-122</td>
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			<td height="21" style="height:21px;width:293px;">Glutamine</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 25030-081</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">N-acetyl cysteine&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# A9165</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Insulin</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# 16634</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Apo-transferrin&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# T1147</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Sodium selenite</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# S-5261</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">DMEM (high glucose)</td>
			<td style="width:317px;">Gibco</td>
			<td style="width:263px;">Cat# 11960-044</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Dapi Fluoromount-G</td>
			<td style="width:317px;">Southern Biotech</td>
			<td style="width:263px;">Cat# 0100-20&#160;</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Poly-D-Lysine&#160;</td>
			<td style="width:317px;">Sigma</td>
			<td style="width:263px;">Cat# A-003-E</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Primaria Plates&#160;</td>
			<td style="width:317px;">VWR</td>
			<td style="width:263px;">Cat# 62406-456</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:293px;">Stock reagents&#160;</td>
			<td style="width:317px;">reconstitution&#160;</td>
			<td style="width:263px;">Concentration used</td>
			<td style="width:192px;">Storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Apo-transferrin</td>
			<td style="width:317px;">10 mg/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">N-acetyl cysteine</td>
			<td style="width:317px;">5 mg/mL in H2O&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Sodium selenite</td>
			<td style="width:317px;">2.5 mg/mL in H2O&#160;</td>
			<td style="width:263px;">1:25,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Collagen IV</td>
			<td style="width:317px;">200 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">TGF-b2</td>
			<td style="width:317px;">20 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:10,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">IL-34</td>
			<td style="width:317px;">100 &#956;g/mL in PBS&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ovine wool cholesterol</td>
			<td style="width:317px;">1.5 mg/mL in 100% ethanol&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Heparan sulfate</td>
			<td style="width:317px;">1 mg/mL in H2O</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:293px;">Oleic acid/Gondoic acid</td>
			<td style="width:317px;">Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol&#160;</td>
			<td style="width:263px;">1:1,000</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Heparin</td>
			<td style="width:317px;">50 mg/mL in PBS&#160;</td>
			<td style="width:263px;">1:100</td>
			<td>-20&#176;C</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:293px;">Solutions&#160;</td>
			<td style="width:317px;">Recipe&#160;</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Comments</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Perfusion Buffer</td>
			<td style="width:317px;">50 &#956;g/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +)</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use when ice-cold</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Douncing Buffer</td>
			<td style="width:317px;">200 &#956;L of 0.4% DNaseI in 50 mL of DPBS++</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use when ice-cold</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Panning Buffer</td>
			<td style="width:317px;">2 mg/mL of peptone from milk solids in DPBS++</td>
			<td style="width:263px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">Microglia Growth Medium (MGM)</td>
			<td style="width:317px;">DMEM/F12 containing 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 2 mM glutamine, 5 &#956;g/mL N-acetyl cysteine, 5 &#956;g/mL insulin, 100 &#956;g/mL apo-transferrin, and 100 ng/mL sodium selenite</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Use ice-cold MGM to pan microglia off of immnopanning dish.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen IV Coating</td>
			<td style="width:317px;">MGM containing 2 &#956;g/mL collagen IV</td>
			<td style="width:263px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Myelin Seperation Buffer</td>
			<td style="width:317px;">9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &#956;L 1 M CaCl2, 5 &#956;L 1 M MgCl2</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Mix well after the addition of&#160; CaCl2 and MgCl2</td>
		</tr>
		<tr height="235">
			<td height="235" style="height:235px;width:293px;">TGF-b2/IL-34/Cholesterol containing growth media (TIC)&#160;</td>
			<td style="width:317px;">MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &#956;g/mL ovine wool cholesterol, 10 &#956;g/mL heparan sulfate, 0.1 &#956;g/ml oleic acid, and 0.001 &#956;g/ml gondoic acid</td>
			<td style="width:263px;"></td>
			<td style="width:192px;">Make sure to add cholesterol to media warmed to 37 &#176;C and do not add more than 1.5 &#956;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.&#160;</td>
		</tr>
	</tbody>
</table>

regeneration of arrayed gold microelectrodes equipped for a real-time cell analyzer

Etiket-gratis celle-baserede assay er fordelagtige for biokemisk undersøgelse på grund af det ikke kræver anvendelse af forsøgsdyr. På grund af dens evne til at yde mere dynamiske oplysninger om celler under fysiologiske tilstande end klassisk biokemiske assays, tiltrækker denne etiket-free real-time celle analyse baseret på princippet om elektrisk impedans mere opmærksomhed i løbet af sidste årti. Dens praktiske udnyttelse kan dog begrænset på grund af relativt dyre udgifter til måling af, hvor dyrt forbrugsmaterialer engangs guld mikrochips bruges til celle analysator. I denne protokol, har vi udviklet en generel strategi for at regenerere klædt guld microelectrodes udstyret til en kommerciel etiket-gratis celle analyzer. Regenereringsprocessen omfatter trypsin fordøjelse, skylning med ethanol og vand og en spinning trin. Den foreslåede metode er testet og vist sig at være effektiv for regenerering og gentagen brug af kommercielle elektroniske plader mindst tre gange, som vil hjælpe forskerne med at spare på de høje løbende omkostninger af real-time celle assays.

Overflade egenskaber af guld mikrochips: regeneration procedurer anvendes i denne protokol blev skitseret i figur 1. Figur 2 viser den mikroskopiske overfladen billeder af friske og regenereret elektroniske plader af optisk mikroskop. Som vist i figur 2A og fig. 2 c, mikroskopiske observationer viste, at der var stort set ingen forskel...

Watch this Scientific Journal Video about Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer at JoVE.com

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

Denne protokol beskriver en generel strategi for at regenerere kommercielle klædt guld microelectrodes udstyret til en etiket-gratis celle analyzer sigter besparelse på de høje løbende omkostninger ofmicrochip-baserede assays. Regenereringsprocessen omfatter trypsin fordøjelse, skylning med ethanol og vand og en spinning skridt, som giver mulighed for gentagen brug af mikrochips.

Regenerering af klædt guld Microelectrodes udstyret til en Real-Time celle Analyzer

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

The overall goal of this procedure is to regenerate commercial electronic plates equipped for a Real-Time Cell Analyzer, or RTCA. This method can help answer key questions in the regeneration of disposable gold-based chips field, including the regeneration of electronic plans used in RTCA. The main advantage of this technique is that the regeneration procedure can be conducted using mild and readily available lab reagents with low toxicity.We first had the idea for this method when we started to use the Real-Time Cell Analyzer which the cost gold-based chips. Begin this procedure with incubation and proliferation of A549 cells in cell-culture dish as described in the text protocol. Add 150 microliters of cell medium to each incubation well of the electronic plate L8.Leave the plate for 30 minutes in the bio-safety hood for equilibration.After 30 minutes, manually insert the electronic plate L8 in the Real-Time Cell Analyzer in the CO2 incubator at 37 degrees Celsius. Next, launch the Real-Time Cell Analyzer operation program on the computer. On the default experiment pattern setup page, select Experiment Model, and name the running assay.On its layout page, select the corresponding wells that are included in the experiment, and input the information such as cell type, number, and drug names in the edit boxes. On its schedule page, add the experimental steps, then select the running time of each step and interval of realtime data acquisition. Click start to measure the background impedance of the media.The experiment file will be saved. The resulting data will be automatically subtracted. On the plot page, add wells, and the graph of time-cell index will be showed.For the cell-proliferation experiment, first click the pause button to pause the program. Then take out the electronic plate, and add 100 microliters of A549 cell suspension buffer per well at room temperature. Leave the plate for 30 minutes in the bio-safety hood to let the cells settle to the bottom of incubation wells.Manually insert the electronic plate in the realtime cell-analyzer station. Then click the start button to continue the experiment. On the plot page, the analyzer continuously records cell index values, or curves, throughout the experiment.When the experiment is complete, enter the plot page, and open the experiment file. Then select all the tested incubation wells and the resulting cell index curves that can be averaged or normalized for the last time before adding pharmaceutical drugs or changing medium to reduce variations between assays. On the data-analysis page, EC50 or IC50 can be calculated.After performing cytotoxicity evaluation of anticancer drug as described in the text protocol, take out the electronic plate containing cells from the Real-Time Cell Analyzer station. Place the used electronic plate in a sterile bio-safety hood at room temperature, and mix the culture medium thoroughly using a pipette. Then use a pipette to aspirate the entire medium.Rinse the electronic plates with 200 microliters of deionized water for three times at room temperature. Digest the remaining cells on the electronic plates with 0.25%freshly-prepared trypsin for one to two hours in an incubator at 37 degrees Celsius. When the digestion is completed, work in the bio-safety hood at room temperature to mix the incubation solution five to 10 times using a pipette, and then remove the solution.Rinse the electronic plates with 200 microliters of deionized water two times. Then wash with 200 microliters of 100%ethanol two times, followed by the same volume of 75%ethanol two times. Finally, rinse with another 200 microliters of deionized water two times.Invert the electronic plate on a sterile gauze or tissue paper to decant the remaining water at room temperature. Then perform ultraviolet sterilization on the regenerated electronic plate for one to two hours in the bio-safety hood at room temperature. After using the plates for drug delivery using mesoporous material as described in the text protocol, digest the remaining cells with trypsin as before.As the pipette is frequently used in the experiments, attention should be taken by the person to avoid scratching of the tip and the surface of the microchips. Rinse the microchips with 200 microliters of deionized water once in the bio-safety hood at room temperature. Then rise the microchips with 200 microliters of absolute ethanol two times at room temperature.Next, rinse the microchips with 200 microliters of 75%ethanol once at room temperature. Add 250 microliters of 75%ethanol in each well, and seal the electronic plate with the sealing film at room temperature. After spinning the electronic plate at 114 times G for two minutes, empty the plate.Invert the electronic plate on a sterile gauze or tissue paper to decant the remaining liquid at room temperature. Repeat the 75%ethanol rinses followed by the spin. Then perform ultraviolet sterilization on the regenerated electronic plate for one to two hours in the bio-safety hood at room temperature.Assess optical characteristics of the regenerated electronic-plate surface by first disassembling the bottom part of the incubation well as described in the text protocol. To evaluate the viability of attached cells after anticancer-drug uptake on the fresh and regenerated electronic plates, use a confocal laser-scanning microscope to monitor the spontaneous fluorescence of DOX molecules absorbed by A549 cells. To record the spontaneous fluorescence of absorbed DOX by the cells, use a 63-times oil objective and 485 nanometers as the excitation wavelength.Next, assess the electrochemical behaviors of regenerated electrodes by first polishing the gold electrode and the glassy carbon electrode to a mirror-like surface with an illuminoslurry on a polishing cloth. Then sonicate the electrodes in water to remove any particles. Next, activate the gold electrode in 0.5-molar sulfuric acid.Obtain Electrochemical Impedance Spectroscopy, or EIS data, of the bare gold electrode and the glassy carbon electrode in 10-millimolar potassium-chloride-electrolyte solution, containing one millimolar potassium pheracyanide. Now, drop 10 microliters of a cell suspension on the surface of the gold electrode in the GCE. After adding the cells, incubate the gold electrode in the GCE in a 37-degree-Celsius incubator for three hours before obtaining EIS data of the electrodes modified with cells, as described in the text protocol.To regenerate the electrodes, immerse the gold electrode in the GCE modified with cells in 0.25%trypsin for 0.5 to two hours. Rinse both electrodes softly with water four times. Then, rinse the electrodes softly with absolute ethanol four times.Next, immerse the gold electrode in the GCE in 75%ethanol for approximately five minutes. After five minutes, rinse the chip softly with 75%ethanol four times before obtaining EIS data of the regenerated electrodes, as described in the text protocol. To evaluate the regeneration efficiency of the arrayed gold electronic plate, microscopic observations were made for both fresh and treated electronic plates.No virtual difference in the optical properties of microchip surface was found. Throescence of doxorubicin molecules is used to assess the cell status on the gold microchips before and after regeneration. The similar homogeneity and well-defined cellular morphology demonstrated the reusability of the chips.The regeneration protocol is applied for the real test of cell proliferation. The growth curves obtained from multiple incubation wells of a tested electronic plate show good consistency, indicating the successful regeneration of the commercial chip. Shown here, the regenerated electronic plate L8 was tested for cytotoxicity evaluation using mesoporous silica materials.The cell index of the mesoporous-materials-treated sample decreased as compared with the control, indicating a slight cytotoxicity of the materials. Further encapsulation and release of the anticancer drug doxorubicin by mesoporous materials caused a dramatic decrease in the growth curve of A549 cells. Once mastered, this technique can be done in four hours if it is performed properly.While attempting this procedure it is important to remember that the type sink digestion time may differ from chip to chip. Following this procedure, other method like confocal laser-scanning microscopy, or even spectroscopy, electrochemical impedance spectroscopy can be performed in order to answer additional questions like assessment of the regeneration effect.

Research

JoVE Journal

Biochemistry

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Meget følsomme og Rapid Fluorescens Detection med en bærbar FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

En Protein Forberedelse Metode til High-throughput Identifikation af proteiner Samspil med en Nuclear Cofactor Brug LC-MS / MS-analyse

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Sporing af DNA-fragmenter i realtid af Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Et fluorescens udsving spektroskopi Assay af Protein-Protein interaktioner på celle-celle kontakter

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

Real-time overvågning af DNA streng Exchange reaktion medieret af Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

Real-time, semi-automatiseret fluorescerende måling af luftvejene overflade flydende pH af primære Human luftvejs epitelial celler

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Overvågning af GPCR-β-arrestin1/2 interaktioner i realtid levende systemer til at fremskynde Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Brug af en ekstracellulær flux analysator til at måle ændringer i Glycolyse og oxidativ fosforylering under mus Sædcapacitation

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

Måling af nukleotidbinding til intakte, funktionelle membranproteiner i realtid

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Aktivering af real-time kompensation i hurtige fotokemiske oxidationer af proteiner til bestemmelse af Protein Topografi Ændringer

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...