This protocol provides a general method to measure the endocytic rate of most plasma membrane proteins by using reducible membrane imp permeate tonation reagents in this procedure, cell surface proteins that contain extracellular lysines or covalently labeled at four degrees Celsius with sulfa N-H-S-S-S biotin shown here in green. Next cells are warmed to 37 degrees Celsius to permit endocytosis of surface proteins. Cells are then returned to four degrees Celsius to stop membrane trafficking and residual surface biotin is stripped using a reducing agent that leaves the disulfide bond.
Thus the only biotinylated proteins that remain are those that originated at the cell surface and were internalized and protected from the stripping procedure. Finally, cells are solubilized and biotinylated proteins are isolated by strep avid and affinity chromatography. Hi, I am Luke Gabriel from Heli Milk Ian's lab in the Department of Psychiatry at University of Massachusetts Medical School.
Today we'll show you a procedure on how to measure endocytic or internalization rates using a reversible biotin elation technique. We use this procedure to measure the internalization rate of dopamine transporter. So let's get started.
One day in advance of the experiment, prepare cells and substrates for use on the following day. Cell types and media will vary, but if experimental cells are not strongly adherent, use an appropriate cell adhesion substrate such as polylysine coated tissue culture plates on each six. Well plate label four wells in total using two separate plates.
For each protein tested, two wells on the control plate will be used for the total surface protein and stripping controls. Prepare a well on the experimental plate for each internalization condition being tested. Plate one times 10 to the six cells in two milliliters of cell culture such that the cells will be approximately 80%confluent by the following day.
After culturing cells, prepare the following solutions and store at four degrees Celsius for use On day two. PBS two plus is prepared from phosphate buffered saline at pH 7.4 and supplemented with 1.5 millimolar magnesium chloride and 0.2 millimolar calcium chloride Tonation quench solution is prepared using PBS two plus supplemented with 100 millimolar glycine NT buffer is made with 150 millimolar sodium chloride one millimolar EDTA 0.2%BSA and 20 millimolar triss at a pH of 8.6 Ripa buffer is prepared using 10 millimolar triss at a pH of 7.4 150 millimolar sodium chloride. One millimolar EDTA 0.1%SDS 1%trite x 101%sodium deoxy coate.
Next, prepare SULF N-H-S-S-S biotin stock solution dissolving in SULFOXIDE or DMSO to a concentration of 200 milligrams per milliliter. Prepare 500 microliter aliquots and store at minus 20 degrees Celsius. Aliquots can undergo several freeze thaw cycles following this tris.
Two carboxy ethyl phosphine hydrochloride or TCEP stock solution is prepared at a concentration of 500 millimolar dissolved in 50 millimolar tris and pH is adjusted to 7.4. Since TCEP is highly acidic, the pH of the solution will need to be adjusted to 7.4 cover TCEP and foil before storing at minus 20 degrees Celsius. On the second day, prepare PBS two plus supplemented with 0.18%glucose and 0.2%IgG protease free bovine serum albumin or PBS two plus GBSA prewarm to 37 degrees Celsius in a water bath.
Thaw out the SFO N-H-S-S-S biotin stock solution at room temperature. Prepare fresh sulfur N-H-S-S-S biotin solution immediately prior to use vortex. The solution vigorously to solubilize the DMSO.
Place both of the plates on an ice bath in a cold room and rinse each well three times with two milliliters of ice cold PBS two plus keeping plates at a slight angle for complete buffer drainage. Following the last wash at 0.75 milliliters per well of the fresh sulfa N-H-S-S-S biotin solution to each. Well incubate for 15 minutes at four degrees Celsius on the ice bath with vigorous shaking on a platform shaker just prior to the end of the incubation.
Prepare another fresh SUHO N-H-S-S-S biotin solution and at 15 minutes replace the old solution with the fresh solution. Incubate for an additional 15 minutes at four degrees Celsius with vigorous shaking on a platform shaker after the second incubation, wash cells three times quickly with two milliliters of quenching solution to quench all non reacting NHS biotin molecules. Then incubate in two milliliters of quenching solution for 15 minutes.
Replace with fresh quenching solution at 15 minutes and incubate for an additional 15 minutes. Keep plates at four degrees Celsius with gentle shaking on a platform shaker for each incubation while the control plate stays in quenching solution on the bench top in the cold room. Remove the experimental plate from the cold room on the ice bath at this point.
If drug treatments are being tested, add appropriate drug concentrations to prewarm PBS two plus GBSA. Here we use either an ethanol vehicle or one MICROMOLAR four ball Myra state acetate or PMA in order to activate the PKC pathway. Wash each well of the experimental plate three times with two milliliters of prewarm PBS two plus GBSA with the appropriate drug treatments added following the last wash.
Add two milliliters of the corresponding PBS two plus GBSA solutions and fill any empty wells with prewarm media to ensure even temperature across the plate. Transfer the cells to a 37 degree Celsius incubator for 10 minutes immediately prior to the end of the incubation. Prepare fresh stripping solution using 50 millimolar TCEP solution in NT buffer and store on ice.
Prepare a sufficient amount for one milliliter per well. Following the 10 minute incubation, quickly transfer the experimental plate to an ice bath and return to the cold room.Rapidly. Wash the experimental wells three times with two milliliters per well of ice cold NT buffer to stop endocytosis.
Then wash the stripping control well three times with two milliliters of ice cold NT buffer. Leave the total control wells in quench solution. Add one milliliter of stripping solution to each experimental and strip control well and incubate on ice for 15 minutes.
Four degrees Celsius with gentle shaking prior to the end of the incubation. Again, prepare the same volume of fresh stripping solution as before. After 15 minutes, replace the old strip solution with fresh solution and incubate an additional 15 minutes four degrees Celsius with gentle shaking to ly the cells first.
Wash all wells that were exposed to stripping solution three times with two milliliters of NT buffer. Then wash all wells including total controls three times with two milliliters of cold pbs. Two plus following the last wash at 300 microliters of RIPPA buffer containing fresh protease inhibitors to each well ly cells by shaking for 20 minutes at four degrees Celsius.
Transfer lysate to 1.5 milliliter micro tubes and clear cellular debris by centrifuge for 10 minutes at 18, 000 GS four degrees Celsius. Use a protein assay compatible with your lysis condition, for example, BCA protein assay from Pierce to determine the lysate protein concentration compared to a standard bovine serum albumin curve. Following this, prepare lysates for strep and affinity chromatography.
Fill 1.5 milliliter micro fuge tubes with equivalent amounts of protein from each sample. In this experiment, we are isolating biotinylated proteins from 50 microgram cell lysates. Add ripa buffer containing protease inhibitors to a final volume of 200 microliters for each sample.
Next vortex, the strep avid and agro beads vigorously before using, using a 200 microliter pipetter with the tip cutoff pipette 20 microliters of stripped avid and beads into each tube. Vortex the tubes and incubate overnight at four degrees Celsius on a tube rotator the following day. Collect the beads by centrifuging the samples at 18, 000 Gs for two minutes at room temperature aspirate off the lysate being careful not to approach the bead pellet with the aspirator add 0.75 milliliters of room temperature.
Rip buffer to each tube and vortex at top speed to wash the beads. Centrifuge at 18, 000 GS for two minutes. Room temperature to collect the beads and repeat this wash vortex centrifuge cycle two more times for a total of three washes after the final wash.
Aspirate as much of the buffer as possible without disturbing the bead pellet. Next at 20 to 25 microliters of two x reducing lamely buffer to each tube. The reducing agent will cleve the N-H-S-S-S biotin dis sulfide bond, releasing the isolated proteins into solution.
As most membrane proteins cannot tolerate boiling incubate tubes on a rotator for 30 minutes at room temperature following reduction and denaturation separate proteins. Using a standard SDS page, gel transfer proteins to a membrane for immuno blotting and blot with an appropriate antibody. In this experiment, we use rat anti DAT antibody from checon number 360 9 at a one to 2000 dilution capture immunoreactive bands using a CCD camera gel documentation system being certain that there are no saturated pixels In this experiment, we use a BioRad versa doc imaging system with quantity one software to quantify each band density.
A representative immuno block from the reversible ation procedure on the dopamine transporter is shown here. The strongest signal is the total amount of surface protein prior to internalization seen in the total lane or T.The strip control or S should ideally be close to blank and display high strip efficiency. The strip efficiency is calculated by using band density measurements in the following formula.
This strip in this experiment was 99.8%efficient strips greater than or equal to 90%Efficiency are optimal and results below this level should be discarded. This example of a pore strip shows a visible band in the strip lane with a strip efficiency of 34%Here the bands of lesser intensity are the internalization lanes from the experimental plate. Cells were treated with either vehicle solution or one micromolar PMA during a 10 minute internalization and dopamine transporter internalization rates were measured.
The internalization rate is calculated using band density measurements as follows, as seen in this graph, 10.4%surface DAT internalized over 10 minutes under vehicle treated conditions. But PMA treatment increased DAT internalization rates to 23.2%of total surface DAT. So you've just seen how to measure plasma membrane protein internalization rates using a reversible bio tonation technique.
It's important to remember two things to freshly prepare the bio tonation reagent as well as the TCP directly before use. If you find you're losing a lot of cells during the protocol, use a cell adhesion substrate on your plates. So that's it.
Thanks for watching and good luck with your experiment.
