standard equipment for horizontal dna electrophoresis can be used . for large numbers of samples ,
we prepare a 20 cm x 24 cm slab with up to four 50-well combs .
make sure the slab does not have scratches , as these can distort the blot image .
create a 1.5% agarose solution ( medium or high gel - strength , low eeo ) in 1x tae .
the volume should be enough to completely submerge the comb teeth -- you will want to load as much sample as possible to maximize detection .
if some agarose solidifies during this step , redissolve using a hot plate and be careful to avoid boiling .
pour the solution into the casting tray . use a comb to rake out any bubbles , as they could later interfere with transfer .
after gel has set , remove combs and place the gel into the gel tank .
completely submerge the gel in 1x tae containing 0.1% sds . for high - throughput analysis of yeast lysates ,
we start with 2-ml cultures grown overnight with rapid agitation in 96-well blocks . in this case , each culture is overexpressing a protein of interest . when analyzing low abundance proteins , larger culture volumes must be used   harvest cells by centrifugation at 2000 rcf for 5 min at room temperature .  
incubate for approximately 30 min at 30c ( you may confirm spheroplasting efficiency by microscopy ) .  
carefully remove supernatant to a new container , e.g. , a 96-well pcr plate .  
if desired , save half of the sample volume and boil it for sds - page analysis . in order to monitor
the extent of transfer later on , include a pre - stained sds - page marker .
additionally , a molecular weight marker consisting of very large proteins ( e.g. , chicken pectoralis extract ) can be used to estimate sizes of the resolved complexes .
run at low voltage (  3 v / cm gel length ) until the dye front reaches ~1 cm from the end of the gel .
it is important that the gel remain cool ; otherwise diffusion of low molecular weight protein ( e.g. , monomers ) can limit their detection . cut a piece of nitrocellulose to the same dimensions as the gel
.   cut 20 pieces of gb004 and 8 pieces of gb002 blotting paper , to the same dimensions as the gel .
cut an additional piece of gb004 to be used as a wick ; make it about 20 centimeters wider than the gel .  
immerse the nitrocellulose , wick , and 4 pieces of gb002 in 1x tbs .   in a plastic container ,
assemble a stack of papers as follows : 20 pieces of dry gb004 , then 4 pieces of dry gb002 , then one piece of pre - wet gb002 .
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 , douse the membrane with tbs as necessary .
if any are present , lift the edge of the gel and reapply buffer until the bubbles can be worked out .
ensure thorough contact between all layers by rolling a pipette firmly across the top of the stack .
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 ( e.g. , a 500 ml bottle of water ) .
spheroplasting solution1.2 m d - sorbitol0.5 mm mgcl220 mm tris , ph 7.550 mm bme ( add fresh)0.5 mg / ml zymolyase 100 t ( add fresh ) lysis buffer100 mm tris 7.5 50 mm nacl 10 mm bme ( add fresh)protease inhibitors ( add fresh)4x sample buffer2x tae 20% glycerol 8% sds bromophenol blue to preference
standard equipment for horizontal dna electrophoresis can be used . for large numbers of samples ,
we prepare a 20 cm x 24 cm slab with up to four 50-well combs .
make sure the slab does not have scratches , as these can distort the blot image .
create a 1.5% agarose solution ( medium or high gel - strength , low eeo ) in 1x tae .
the volume should be enough to completely submerge the comb teeth -- you will want to load as much sample as possible to maximize detection .
if some agarose solidifies during this step , redissolve using a hot plate and be careful to avoid boiling .
pour the solution into the casting tray . use a comb to rake out any bubbles , as they could later interfere with transfer .
after gel has set , remove combs and place the gel into the gel tank .
for high - throughput analysis of yeast lysates , we start with 2-ml cultures grown overnight with rapid agitation in 96-well blocks . in this case , each culture is overexpressing a protein of interest . when analyzing low abundance proteins , larger culture volumes must be used   harvest cells by centrifugation at 2000 rcf for 5 min at room temperature .  
incubate for approximately 30 min at 30c ( you may confirm spheroplasting efficiency by microscopy ) .  
carefully remove supernatant to a new container , e.g. , a 96-well pcr plate .  
if desired , save half of the sample volume and boil it for sds - page analysis . in order to monitor
the extent of transfer later on , include a pre - stained sds - page marker .
additionally , a molecular weight marker consisting of very large proteins ( e.g. , chicken pectoralis extract ) can be used to estimate sizes of the resolved complexes .
run at low voltage (  3 v / cm gel length ) until the dye front reaches ~1 cm from the end of the gel .
it is important that the gel remain cool ; otherwise diffusion of low molecular weight protein ( e.g. , monomers ) can limit their detection .
cut 20 pieces of gb004 and 8 pieces of gb002 blotting paper , to the same dimensions as the gel .
cut an additional piece of gb004 to be used as a wick ; make it about 20 centimeters wider than the gel .  
immerse the nitrocellulose , wick , and 4 pieces of gb002 in 1x tbs .   in a plastic container ,
assemble a stack of papers as follows : 20 pieces of dry gb004 , then 4 pieces of dry gb002 , then one piece of pre - wet gb002 .
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 , douse the membrane with tbs as necessary .
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 gb002 pieces on top of the gel .
ensure thorough contact between all layers by rolling a pipette firmly across the top of the stack .
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 ( e.g. , a 500 ml bottle of water ) .
spheroplasting solution1.2 m d - sorbitol0.5 mm mgcl220 mm tris , ph 7.550 mm bme ( add fresh)0.5 mg / ml zymolyase 100 t ( add fresh ) lysis buffer100 mm tris 7.5 50 mm nacl 10 mm bme ( add fresh)protease inhibitors ( add fresh)4x sample buffer2x tae 20% glycerol 8% sds bromophenol blue to preference
sdd - age was first reported by kryndushkin et al . , to study sds - resistant complexes of the [ psi+ ] prion in yeast , and has since found widespread use studying both prion and non - prion aggregates . however , transfer of the proteins to a membrane following electrophoresis in an agarose gel is problematic , and can result in a distorted blot image . additionally , the submerged electroblotting technique most commonly used introduces practical limitations for the size of the gel and thus the number of samples that can be processed .
we have addressed these problems by employing downward capillary transfer , a simple procedure which uses a stack of dry blotting papers to transfer proteins from the gel to a nitrocellulose membrane .
there are a few things to consider before using sdd - age . for crude samples ( e.g. , lysates ) ,
sdd - age does not fully denature the protein complexes of interest , so the protein(s ) to be detected must bear an epitope tag outside of the amyloidogenic region .
lysates can generally be prepared as they would be for a normal sds - page , with two important differences .
the partially denaturing conditions used here are not sufficient to inactivate proteases , and can also make target proteins more susceptible to proteolysis . use a complete protease inhibitor cocktail at at least two - fold the recommended concentration .
second , heating the samples should be avoided . if an all - monomer negative control is desired , for instance to confirm that high - molecular - weight species are not due to covalent modifications , a 10-minute incubation at 95c can be used , which will restore most amyloids to monomeric protein .
 amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions , which are conformationally self - templating proteins that are thought to have beneficial roles in lower organisms . 
 amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity . 
 however , resolution of amyloid polymers based on size and detergent insolubility has been made possible by semi - denaturing detergent - agarose gel electrophoresis ( sdd - age ) . 
 this technique is finding widespread use for the detection and characterization of amyloid conformational variants . here , we demonstrate an adaptation of this technique that facilitates its use in large - scale applications , such as screens for novel prions and other amyloidogenic proteins . 
 the new sdd - age method uses capillary transfer for greater reliability and ease of use , and allows any sized gel to be accomodated . 
 thus , a large number of samples , prepared from cells or purified proteins , can be processed simultaneously for the presence of sds - insoluble conformers of tagged proteins . 
Protocol
Part 1: Preparing the gel
Part 2: Preparing samples
Part 3: Transfer
Discussion