Hello, I'm Randall Hoffman, a graduate student in Susan Linus Laboratory in the biology department at MIT. Today I'm going to introduce you to semi denaturing detergent agro rose, gel electrophoresis, or SDD age. Our lab uses this technique to screen for prions and other alogenic proteins.
Our version of SDD age is simple and reliable and can be scaled to accommodate any number of samples. Amyloids are exceptionally resistant to denaturation by SDS, which allows them to be separated from other proteins using SDDH. However, data from SDDH cannot by itself be used to confirm that something is amyloid.
To do that, you need to use other techniques. With that in mind, let me show you. S-D-D-H-S-D-D-H can be performed using standard equipment for horizontal DNA electrophoresis.
First, you need to assemble the gel casting tray. The size of the apparatus will depend on how many samples you have. For a large number of samples, we use a 20 by 24 centimeter slab, and up to four 50 well combes.
To prepare the gel, you'll need medium or high gel strength low, EEO agros, one XTAE buffer, and a 10%stock solution of SDS Make a 1.5%AROS solution and one XTAE. You'll want to make the gel as thick as possible so you can load the most sample and maximize detection. Heat the mixture in the microwave until the aros is completely dissolved.
Quickly add the 100 XSDS stock to 0.1%and swirl to mix. If some agro solidifies during this step, red, dissolve it on a hot plate. Be careful not to let it boil.
After adding SDS as the solution will easily bubble over, pour this solution into the casting tray while it is still hot. Then use a comb to get rid of any bubbles which may interfere later on during gel transfer. After the gel is completely cooled and solidified, carefully remove the combs and place the gel into the tank.
Completely submerge the gel in one XTAE with 0.1%SDS. You are now ready to load your samples. The protein samples used in SDDH are prepared just like in an SDS page with two important exceptions.
First, you should avoid heating the samples which could denature the aggregates into protein monomers. Second, take care to avoid protein degradation. Because the denaturing conditions used here are not sufficient to inactivate all proteases, we recommend using at least twice the recommended concentration of a complete protease inhibitor cocktail.
In this demonstration, we'll use lysates from yeast cells that are over expressing a particular protein of interest. We generally use two mil overnight. Cultures grown in 96, well blocks available from VWR, but when analyzing low abundance proteins, larger culture volumes must be used.
Harvest the cells by centrifuging the culture at 2000 RCF for five minutes at room temperature following centrifugation, remove the supra natant and wash the cells by Resus suspending in water and centrifuging again after the second spin, remove the supernatant and resin the cells in spare plating solution. The zy in the solution digest the cell walls, which will allow you to ly the cells efficiently and a small volume of lysis buffer. Incubate for 30 minutes at 30 degrees Celsius.
Pellet the spare plastic cells this time at 800 RCF for five minutes. Remove the sate completely and resuspend each pellet and 100 microliters of lysis buffer. Next, cover the block with tape and vortex on high speed for two minutes.
This helps all the sphere plastics to LY P out the cellular debris at 4, 000 RCF. For two minutes. Carefully transfer the S natin to a 96 well PCR plate.
Typically, we analyze hundreds of samples at a time in our laboratory, so to assist us with this is a liquid handling system. If desired, determine the protein concentration of the lysates. Next, add four x sample buffer to the samples to generate lysates containing one x sample buffer incubate for five minutes at room temperature.
Now load the samples into the gel. If desired, save half of the samples for SDS page analysis. Also, load molecular weight markers, including prestained markers and a very high molecular weight marker such as chicken flector s extract.
If desired, it is important to keep the gel cool to prevent diffusion of low molecular weight proteins, so you may want to run it in the cold room. Run the gel at low voltage less than three volts per centimeter of the gel length. Run the gel for several hours until the diare reaches about one centimeter from the end of the gel.
Because amyloid is not denatured by 2%SDS at room temperature, it will remain intact while other complexes, including most non amyloid aggregates, dissociate in the presence of 2%SDS. Thus, most proteins will be monomeric and run quickly through the gel. While the SDS resistant high molecular weight amyloids will migrate more slowly.
After this, the gel will be ready for transfer. Although multiple transfer methods can be used, we obtain the best results using capillary transfer. Since capillary transfer can accommodate any size of gel, it is practical to analyze hundreds of samples in capillary transfer.
A stack of dry blotting papers acts to draw buffer through the gel depositing proteins onto a nitrocellulose membrane placed between the papers and the gel. You'll need to cut a piece of nitrocellulose, such as high bond C, extra to the same dimensions as the gel. You'll also need to cut 20 pieces of GBO oh four and eight pieces of GBO oh two blotting paper to the same size as the gel.
GB oh four is very thick and absorbent. While GBO oh two is much thinner and has a finer texture, which ensures more even contact with the membrane. Cut an additional piece of GBO oh four to be used as a wick and make it about 20 centimeters wider than the gel immersed.
The nitrous UL WIC and four pieces of GBO oh two and one XTBS in a plastic container assemble a stack of papers as follows, 20 pieces of dry GBO oh four. Then four pieces of dry GBO oh two. Then one piece of pre wetted GBO oh two.
Lay the nitrocellulose on top of this stack. Rinse the gel on the casting tray briefly in water to remove excess running buffer. Then carefully begin to slide the gel off the tray onto the stack while sliding the gel off the tray dows the membrane with TBS as necessary.
The extra buffer helps prevent bubbles from becoming trapped into the gel. The transfer pipette works well for this purpose after the gel has been completely moved to the stack. Check thoroughly for bubbles, if any are present.
Lift the edge of the gel and reapply buffer until the bubbles can be worked out. Put the remaining three pre wetted GBO oh two pieces on top of the gel. Ensure thorough contact between all layers by rolling a pipette firmly across the top of the stack.
Flank the transfer stack with two elevated trays containing TBS drape the pre-wet wick across the stack, such that either end of the wick is submerged. In TBS cover the assembled transfer stack with an additional plastic tray bearing extra weight such as a 500 mil bottle of water. The wick will draw a buffer from the trays and the dry blotting papers will draw the buffer through the gel over the course of several hours.
This will deposit the proteins onto the membrane, allow the transfer to proceed for a minimum of three hours or overnight. After the capillary transfer is complete, the membrane can be processed by standard western blotting. Here you see a typical blot of proteins that were over expressed in a yeast strain that predisposes certain proteins to form amyloid.
You can clearly see that some of our candidates form amyloid or high molecular weight SDS resistant species very well like those shown here and here. Others, however, do not form aggregates and are completely monomeric such as these and these. Additionally, you can see that the size of the amyloid polymers differs depending on the protein, which you can see in these two lanes.
We've just showed you a simple method for analyzing high molecular weight, insoluble aggregates, such as amyloids of candidate prions. We hope you've enjoyed watching our presentation and find this technique useful and studies of Amy immunogenic proteins. Thanks for watching and good luck with your research.
