Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

This vertical presents important steps in assembling cell membrane affinity chromatography columns with functional transmembrane receptors. These columns can be used for the identification of small molecules present in complex extracts and interacting with immobilized transmembrane receptors. Cellular membrane affinity chromatography columns speed up the identification of biologically active compounds present in complex mixtures.

The use of cellular membrane affinity chromatography columns can be of particular interest to those interested in the identification of pharmacologically active compounds present in complex natural matrices, such as plant, fungal, or bacterial extracts. To begin, prepare 200 millimolar benzamidine stock solution by dissolving 120 of benzamidine in five milliliters of ultrapure deionized water. Prepare a 10 millimolar PMSF stock solution by dissolving 0.017 grams of PMSF in 10 milliliters of ethanol in a fume hood.

Prepare 100X proteases inhibitor cocktail by dissolving commercially available protease inhibitor mixture in one milliliter of ultrapure deionized water. Mix thoroughly and aliquot 200 to 300 microliters of the cocktail. Prepare 100 millimolar ATP stock solution by dissolving 55.114 milligrams of ATP disodium salt hydrate in one milliliter of the ionized ultrapure water.

Mix thoroughly and aliquot 100 microliters of the mixture. Prepare buffer by dissolving 3.03 grams of Tris-HCL, 2.9 grams of sodium chloride, 0.22 grams of calcium chloride anhydrous, 0.2 grams of magnesium chloride hexahydrate, and 0.19 grams of potassium chloride in 500 milliliters of ultrapure deionized water. Prepare homogenization buffer by mixing 17.3 milliliters of the Tris buffer with 0.3 milliliters of benzamidine stock solution, 0.2 milliliters of PMSF stock solution, 0.2 milliliters of proteases inhibitor cocktail, 20 microliters of ATP stock solution, two milliliters of glycerol, and 0.029 grams of EDTA.

Adjust pH to 7.4 using hydrochloric acid solution. Spin down the cells harvested at four degrees Celsius for five minutes at 400 x g and remove the supernatant and wash the remaining cell pellet with 10 milliliters of 1X pH 7.4 ice cold PBS. Discard the supernatant and add 20 milliliters of homogenization buffer.

Place the conical tube on ice. Transfer the cell suspension into a 40 milliliter Dounce homogenizer tissue grinder, homogenize the suspension manually on ice using 40 up and down pestle strokes. Transfer homogenized cell suspension into a 50 milliliter conical tube, discard the pellet and transfer the supernatant to a new conical tube.

Save the cell membrane pellet and discard the supernatant. Prepare solubilization buffer by mixing 8.7 milliliters of the buffer solution with 0.1 milliliters of PMSF stock solution, 0.15 milliliters of benzamidine stock solution, 0.1 milliliters of proteases inhibitor cocktail, 10 microliters of ATP stock solution, one milliliter of glycerol, and 0.2 grams of sodium cholate. Transfer the solubilization buffer into the conical tube with cell membrane fragments.

And resuspend the pellet. Rotate the resulting mixture at 150 RPM at four degrees Celsius for 18 hours. After 18 hours, centrifuge the solubilization mixture at four degrees Celsius for 30 minutes at 47, 900 x g.

Add 100 milligrams of IAM. PC.DD2 particles to the supernatant and rotate the resulting suspension mixture at 150 RPM at four degrees Celsius for one hour. Prepare dialysis buffer in four liters of ultrapure deionized water by adding 24 grams of Tris-HCl, 23.4 grams of sodium chloride, 0.06 grams of calcium chloride, 5.85 grams of EDTA, and 0.07 grams of PMSF.

Dissolve PMSF first in a small volume of ethanol and then add slowly into the beaker with the buffer. Cut 10 centimeters of cellulose membrane dialysis tubing to prepare dialysis tube, and transfer the suspension containing IAM. PC.DD2 particles and cell membrane fragments into the dialysis tube.

Close both ends of the dialysis tube with dialysis tubing clips. Place the dialysis tube in the dialysis buffer. After 24 hours, place the dialysis tubing in the freshly prepared dialysis buffer and continued dialysis for another 24 hours.

Prepare 10 millimolar pH 7.4 ammonium acetate buffer that will be used to wash the IAM. PC.DD2 particles and in column characterization experiments by dissolving 0.778 grams of ammonium acetate in one liter of ultrapure deionized water. After 48 hours of dialysis, remove the dialysis tube from the dialysis buffer and transfer the content of the dialysis tube into a 15 milliliter conical tube.

Discard the supernatant and wash the remaining pellet three times with 10 milliliters of ammonium acetate buffer. After the third wash, resuspend the remaining pellet in one milliliter of ammonium acetate buffer. Mix it thoroughly and use the resulting slurry to pack the 5 x 20 glass column to yield the CMAC chromatography column.

To pack the column, place a bottom filter previously soaked with ammonium acetate buffer into the filter holder. Fit the filter holder in the glass column and screw the column cap to secure the holder's position. Place the column vertically in a finger clamp and secure it in the lab stand.

Place a beaker below the column, transfer a small volume of the slurry into the glass column slowly with a single channel pipetter holding the pipette tip against the glass column wall. To speed up the packing process, remove the buffer from above the stationary bed with a micropipette between each step. Place a top filter and screw the adapter unit so that there is no remaining buffer above the stationary phase.

Secure the position of the adapter unit with the adapter lock. Connect the column to a high performance liquid chromatography pump, and to wash the column overnight with ammonium acetate buffer, set the flow rate to 0.2 milliliters per minute. For longer storage, run the column with 0.05%sodium azide solution in ammonium acetate buffer, wearing a lab coat, safety glasses, and gloves when working with sodium azide and store at four degrees Celsius.IAM.

PC.DD2 particles with immobilized neuroblastoma TrkB cell membrane fragments and in the presence of BDNF resulted in fluorescent particles. No fluorescence was observed in TrkB null cell membrane encapsulated IAM particles or when no BDNF was used as in TrkB cell membrane encapsulated IAM particles. A functional CMAC columns typical frontal affinity chromatogram of increasing 7, 8-DHF concentrations of one millimolar, 750 nanomolar, 500 nanomolar, and 300 nanomolar to the immobilized TrkB receptors are shown, which showed a concentration dependent change in retention time.

A nonfunctional CMAC column showed the lack of concentration dependent changes in retention of the marker ligand. A CMAC negative control column was prepared by immobilizing cell membrane fragments, not expressing the targeted protein to rule out non-specific interactions. The addition of 0.2%aqueous Gotu kola extract resulted in a significant reduction of 7, 8-DHF retention, indicating the presence of competing ligands for the agonist binding site.

The lack of reduction of 7, 8-DHF retention on the CMAC negative TrkB null column further confirmed the lack of functional TrkB receptors on this column. The missing peak chromatography approach identified a compound strongly retained on TrkB column, while eluting early on TrkB null column, indicating specific interaction. Its crucial to know your source of receptors for fast cellular membrane affinity chromatography column.

Make sure to check the literature to properly design homogenization and solubilization buffers. Cellular membrane affinity chromatography column prepared in this protocol was used to identify new potential drug hits and to characterize the nature of the interaction between immobilized receptor and its natural ligands.