באמצעות מערכת GELFREE 8100 ההיפוך חלוקה על ההיפוך חלוקה משקל מבוסס מולקולרית עם שלב החילוץ נוזלי

Hi, my name is Jay Harkins. I’m a research scientist at Protein Discovery. Today I will demonstrate how easy it is to use the new gel free 8, 100 Fractionation system to achieve high recovery molecular weight based fractionation.

Using gel free, you can load up to one milligram of total protein per channel, and reproducibly fractionate up to eight samples in about 90 minutes. So the system includes an instrument, disposable cartridge, and all the buffers you need to perform the experiment. So let’s get started.

Begin this procedure by setting up and labeling up to eight 500 microliter tubes to each tube. Add up to one milligram of the protein sample in a volume of up to 112 microliters. The amount of protein in the sample depends on the complexity of the sample.

For more complex samples, such as the tissue homogenate used for this demonstration, 150 to 200 micrograms per channel will generate the best results. Next, add 30 microliters of five x sample buffer, which is provided in the gel free cartridge kit. To each protein sample, add eight microliters of one molar DTT.

Then adjust each sample with water to a final volume of 150 microliters. Then heat the vials for five minutes at 95 degrees Celsius to denature the sample. Allow the heated samples to cool to room temperature while the sample’s cool.

Remove the gel free 8, 100 cartridge from the foil pouch. Remove and discard the plate sealer. Remove the storage buffer from the cartridge compartments using a pipette.

If all eight chambers of the cartridge will be used, invert the cartridge to drain the storage buffer from the compartments. Next eight milliliters of gel free running buffer is added to each of the anode buffer reservoirs. Six milliliters is added to each of the cathode buffer reservoirs, and 100 microliters is pipetted into each of the collection chambers.

Using an eight channel 100 to 200 microliter pipetter, remove and discard any buffer that flowed from the cathode buffer reservoir into the sample loading chamber and immediately load the samples into the loading chambers. Once the samples have been loaded, place the cartridge into the gel free 8, 100 fractionation station. Then lower the electrode arrays and close the lid using the easy to program graphical user interface on the instruments touchscreen display, go to the list of available pre-programmed methods.

Select the appropriate method by pressing the method button. Press the retrieve button and enter the number of the desired pre-programmed method Using the onscreen keypad press okay. Then press the apply button.

The method that has been applied will appear at the bottom of the main screen. Each of the pre-programmed methods consists of set voltages and time-based pauses designed to resolve proteins within a specific range of molecular weights. Once the appropriate method has been selected, press the channel button on the main screen.

Then select the channels that will be used in the run. To select all eight channels, press select all. Then press done.

To return to the main screen, ensure that the safety lid is closed and that the indicator light is green at the bottom of the screen. Now press start to begin the experiment while the system is running, the circles shown in the box on the left side of the screen depict the status of each channel. A green circle indicates that the channel is active.

The applied voltages and currents are displayed to the right of the channel status circles. The gel free 8, 100 will automatically pause for fraction collection at the interval specified in the method. When the instrument is paused to collect fractions, a text box will appear notifying you that a pause has occurred and the status circles will turn yellow.

Inside the cartridge, a constant voltage is applied between the anode and cathode reservoirs. Under this potential, the protein mixture is electrophoretic driven from the loading chamber and into the specially designed precision cast. Gel proteins first stack up inside a sharp band in the stacking gel and then resolved based on their respective molecular weights in the resolving gel.

As proteins elute from the gel, they are trapped and concentrated in liquid phase. In the collection chamber gel free. The instrument is then paused.

Based on preselected time intervals and fractions are collected using a pipette. Then the potential is applied again to allude to end entrap the second molecular weight fraction of interest. This process is then repeated until all desired fractions have been collected.

Each cartridge contains eight independent electrophoretic channels so the user can run as many channels as they need and save the unused channels for use at a later date. To remove the fractions, open the lid of the instrument, then use the innate channel pipette. Transfer the 150 microliters of liquid in each of the collection chambers to collection tubes or a multi-well plate.

Once the fractions have been removed, wash the collection chamber twice with gel free running buffer by adding 100 microliters per channel and pipetting up and down twice. After washing, add 100 microliters of gel free running buffer back into the collection chambers. Close the lid and press the resume button.

The gel free 8, 100 will run until the next time interval. The total time remaining in the experiment is shown on the right hand side of the screen. Press the button above the box displaying time to toggle between time remaining time elapsed, and time to pause.

Once all of the fractions have been collected, they can immediately be used for further downstream preparation, such as isoelectric focusing or gel electrophoresis or for analysis using liquid chromatography, mass spectrometry or immuno affinity. In this demonstration, eight protein samples at 200 micrograms each were partitioned into 12 fractions each for a total of 96 fractions. To visualize the results of the fractionation and aliquot of each of the 12 fractions from the bovine liver homogenate sample was then run on a standard 1D gel and silver stained.

As shown in this figure, the protein sample was fractionated into 12 distinct fractions spanning the molecular weight range from 3.5 kilodaltons to 150 kilodaltons. The gel shown here highlights the ability of the gel free 8, 100 system to fractionate a large amount of protein into distinct molecular weight fractions. This is an incredibly powerful tool for top-down and bottom-up proteomics using mass spectrometry as well as immuno affinity experiments where a single purified intact protein molecular weight fraction is required.

It’s been a pleasure showing you how easy it is to use the gel free 8, 100 fractionation system to reproducibly partition your sample into discrete molecular weight fractions, and achieve high liquid phase recovery. So that’s it. Thanks for watching and good luck with your experiments.