MATERIALS AND METHODS

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MATERIALSAND METHODS

Sizeexclusion column preparation.A particle mask was obtained and labeled appropriately using the labtape. A 1.5 x 50cm Bio-Rad column was affixed to the ringstand byusing two secure clamps while ensuring that the column remainedperfectly vertical. A stopcock was attached at the bottom of thecolumn and turned to ‘closed’ position. An approximately 2.5mLdegassed elution buffer (dg-EB) was added to the column usingtransfer pipette. A 100ml Sephadex G-75 solution, that was alreadyprepared and degassed, was measured using a 150ml beaker. Back intothe column, the hydrated Sephadex was swirled to enhance thehomogeneity while checking the viscosity. The viscosity wasstandardized using 10mL dg-EB increments to a ‘pourable’ state.The column was filled with Sephadex solution through pouring in aone-fluid-motion while ensuring that no air bubbles were in thecolumn. The particle mask was set aside temporarily by moving thebeaker and all the other things that touched Sephadex into the hood.The Gel was allowed to settle completely in the column while keepingthe stopcock open. During this time, the effluent was collected intoa 50mL beaker, and a 20mL syringe pump used in increasing thecolumn`s flow rate. The effluent was collected from the column untilthe meniscus was just above the gel column`s top, while ensuring thatcolumns remained at approximately 40cm height. In the currentexperiment, the column was 39.5cm tall, and this height wasstandardized by removing and adding the hydrated Sephadex using thetransfer pipette. After the column settled completely, about 5mLdg-EB effluent was added onto the top of the column for storagepurposes, with the gel resin being at a perfect level. Finally, thebeaker and everything else that touched the Sephadex were washedusing di-H2Ointo a waste container.

Preparationof Column Elution Protocol.A 250ml Nalgene filter apparatus was obtained and opened to help inplacing the magnetic stirrer into the collection vessel and then seton stirplate. Thereafter, the apparatus was connected to a vacuumline using an appropriate tubing and reconnected to the filtermodule. The filter cover was simultaneously removed by pouringapproximately 150-120mL EB onto the filter in order to initiate avacuum through aspiration. The filter cover was securely replaced andstirring initiated for 30min. Finally, seventy-two-13 x 100mm testtubes were marked at 1mL mark, labeled and stored for later use. Fullabsorption spectrum was obtained for each of standard solutions. Thevalues obtained during the experiment were Blue dextran 616.45nm(unique) and 388nm, DNP-Pro was 399.1nm while unknown protein was545.2nm (unique) and 408.4nm.

GelFiltration Protocol.The dimension of the completely settled column was recorded as40.7cm. The stopcock was opened, and the buffer that was above thecolumn drained using a transfer pipette into beaker ‘A’. About1mL of the separation mixture was added onto the column using atransfer pipette. An empty 50mL beaker, (beaker B), was placed belowthe column, and the separation mixture was run into the column untilall the meniscus was above the resin level. An approximate 1mL dg-EBwas added carefully onto the column top using a transfer pipetteafter the separation mixture had been fully absorbed onto the column.The stopcock was opened further to continue collecting the effluentinto the beaker B until the meniscus was above the resin level. Theaddition of 1mL dg-EB and collection of the effluent was repeatedlycarried out until the separation mixture loaded completely and safelyonto the gel resin. Thereafter, the column top was filled with dg-EBas the effluent continued to collect in beaker ‘B’ and testtubes. This was done in two main stages. In the first stage, effluentwas left to run until the blue band was at approximately 10cm fromthe column bottom. The volume of the effluent recorded aftercompletion of elution was 19ml. The second stage involved collectingthe effluent into the 1mL marked test tubes until all the colorseluted completely from the column. This step required the column tobe constantly refilled with dg-EB. After complete elution of all thecolored components from the column, the SpectroVis Plus wascalibrated on a blank (pre-lab TBD) sample. The 1.5mL di-H2Owas added to each of the 72 testtube-fractions containing theeffluent. Thereafter, the entire mixture in the test tube (effluentand di-H2O)was transferred into plastic cuvette, and complete absorbancespectrum for the 72 samples were recorded and saved in a single file.The Blue Dextran Eluant had an absorbance of 3-7, unknown Fractionrecorded 19-24 while the DNP Pro had an absorbance of 30-49.

Reference

ChromatographFigure