The overall goal of the following experiment is to characterize a MultiPro complex from cellular lysates using blue native poly acrylamide gel electrophoresis, which in contrast to SDS page, allows protein separation under native conditions and preserves protein protein interactions. This is achieved by dialyzing the cellular lysate to remove excess salts making the sample applicable to separation by blue native page as a second step. The lysate is applied to first dimension blue native page, which separates protein complexes under native conditions according to their size and shape.
Next, a second dimension. Denaturing SDS page is performed in order to subdivide the complexes into their individual constituents. Proteins can be visualized by silver or kumasi staining or western blotting.
Here separated proteins were detected by silver stain in the following demonstration, however, western blotting will be used. Hi, I'm Brita Bloom from the laboratory of Wolfgang Sharmel in the Department of Molecular Immunology at the Max Plank Institute of Immunobiology in f Fryberg Germany. Hi, I'm Dina Fiala.
I'm also from the Sham Lab. Today we will show you a procedure for the analysis of MultiPro complexes from cellular lysates. We use this procedure in our laboratory to study the composition of multis subunit membrane receptors.
However, in this video we use tic proteasome as an example. So let's get started. Let's Prior to the start of this procedure, prepare all buffers as described in the written protocol and keep them at four degrees Celsius.
Harvest 10 million HEC 2 9 3 cells and pellet them by centrifugation at 350 times G for five minutes at four degrees Celsius. Then wash the cell pellets with one milliliter of ice cold PBS and use centrifugation to pellet the washed cells. Repeat this wash twice, then resuspend the washed cell pellet in 250 microliters of ice.
Cold blue, native lysis, buffer, and incubate the suspension on ice for 15 minutes. Following incubation, centrifuge the life cells at 13, 000 times G for 15 minutes at four degrees Celsius to remove insoluble material. Meanwhile, melt a hole in the cap of a 1.5 milliliter micro centrifuge tube using the heated large diameter side of a pasta pipette.
Then place the tube on ice to cool down to four degrees Celsius. Once centrifugation is complete, transfer the lysate to the tube for salt removal by dialysis. Place a 10 kilodalton cutoff dialysis membrane on top of the open tube.
Close the cap and ss off any excess dialysis membrane that sticks out. Then carefully seal the side of the cap with param once sealed, invert the tube and place it upside down into a 50 milliliter conical tube. Centrifuge the sample at the lowest possible speed for 10 seconds.
In a cell culture centrifuge, prepare a 100 milliliter beaker with cold blue native dialysis buffer and a magnetic stir using at least 10 milliliters of blue native dialysis buffer per 100 microliters of sample. Then remove the inverted tube from the conical tube using tweezers. To avoid turning the tube right side up.
Aix the tube with tape upside down inside the beaker and remove any air bubbles from the hole beneath the cap. Using a bent past pipette switch on the magnetic stir and leave the sample for six hours or overnight in the cold room to dialyze. Check the sample occasionally to ensure that stirring is not creating air bubbles at the dialysis membrane.
Following incubation, transfer the dialyze cell lysate into a new chilled micro centrifuge tube and leave it on ice until loading of the gel gradient. Gel pouring is done at room temperature with a gradient mixer. Place the gradient mixer on a stir plate and attach it to a piece of flexible tubing.
Close the channel of the gradient mixer using the valve and close the tubing with a clamp. Place a magnetic stir into the cylinder connected to the tubing. Next, thread the flexible tubing into a peristaltic pump and attach a syringe needle to its end.
Then place the needle between the two glass plates at the top of the gel apparatus. Prepare 4%and 15%separating gel solutions so that the combined volumes are equal to the volume at the separating gel. Add a PS and TM me immediately before.
Use and pour the gel solutions into the corresponding cylinders of the gradient mixer. Then switch on the and open the valve. Press over the left cylinder to force out the air bubble inside the channel connecting the two gel reservoirs.
Switch the pump on at five milliliters per minute. Remove the clamp and allow the gel to slowly flow between the glass plates. Slowly raise the needle as the gel pauses, ensuring that the needle is always above the liquid.
Allow all the liquid to enter the gel apparatus. Then overlay the liquid gently with isopropanol and rinse the apparatus with distilled water. Allow the gel to polymerize for at least 30 minutes at room temperature.
Next, prepare a 3.2%stacking gel, adding a PS and TM me immediately before use. Pour the stacking gel on top of the separating gel and introduce the comb between the glass plates, avoiding bubbles. After the stacking gel has polymerized, cool the gel down to four degrees Celsius immediately before sample loading.
Remove the comb slowly pulling it out at an angle to the plane of the gel. This allows air to enter the pockets rapidly, which improves the quality of the wells to separate the dialyze cell lysate by blue native page load 20 microliters of the ferritin marker and 40 microliters of the dialyze lysate to the dry wells At four degrees Celsius, empty lanes are filled with equal volumes of dialysis buffer. Then overlay the samples in each well with cold cathode buffer.
Fill the inner chamber with cold cathode buffer and the alsa chamber with cold anode buffer. Work at four degrees Celsius and apply 100 volts until the samples have entered the separating gel. Then increase the voltage to 180 volts and run the gel until the die front reaches the end of the gel.
The run takes three to four hours. For second dimension SDS page, prepare a standard 10%SDS gel with a single large lane. One lane for the molecular weight marker and one lane for an aliquot of the dialyzed lysate control that has been mixed with SDS sample buffer and boiled for five minutes at 95 degrees Celsius, remove the blue native page gel in the plates from the electrophoresis apparatus and gently pry up one plate.
Remove the stacking gel and cut out the lane of the blue native page gel containing the proteins of interest. Place the blue native page gel slice in SDS sample, buffer and incubate for 10 minutes of room temperature on a shaker following incubation. Boil the gel slice briefly in a microwave, then incubate for another 15 minutes.
In the SDS sample buffer on a shaker, remove the comb of the SDS gel and fill some SDS buffer into the well load the blue native page gel slice into the large well of the SDS gel. Avoiding air bubbles. You may want to use a tweezer for help as the slice of gel has to be exactly on the stacking gel.
Overlay the slice with SDS sample buffer, then load the marker and the lysate control. Perform electrophoresis and protein transfer according to standard protocols. To interpret the results, it is important to know that the position of a protein in the second dimension blue native SDS page gives evidence whether the protein is part of a MultiPro complex or not.
Monomeric proteins will migrate in a hyperbolic diagonal due to the gradient gel in the first dimension, and as a linear gel in the second dimension, subunits of MultiPro complexes will be found below the diagonal forming a vertical line. If a certain protein is part of distinct complexes, several spots are detected in a horizontal line. In summary, the two dimensional blue native page SDS page approach allows determination of MultiPro complex composition size and the relative abundance.
In this demonstration, several proteasome or subunits were detected by western blotting. The subunits beta two and MCP 21 could be detected as individual spots which are vertically arranged indicating that they are a part of the same complexes. PA 28 is found in one vertical line with beta two and CP 21 and thus part of the same complex.
When looking at the horizontal lines, one can see that beta two and MCP 21 are part of three distinct complexes. PA 28 is only found in one of those complexes. Therefore, these MultiPro complexes could be identified as the 20 success proteasome the 20 s proteasome.
Together with P 28 and the 20 S proteasome alone, We've just shown you how to separate and characterize native MultiPro complexes from a cellular lysate using blue native page. When doing this procedure, it's important to remember to carefully dialyze the sample. To avoid contact with SDS and to always work at four degree, we recommend comparing different detergents for cellular lysis to achieve both integrity of a motor, pretty complexes and best solubilization.
So that's it. Thanks for watching and good luck with your experiments. I'll in.
