Dr. Proctor: Good morning, everyone. Thank you for joining us for this Simple Western platform introduction

I'd like to introduce you to Simon and Sally. Both are the latest in immunoassay technology, Simon delivering 12 automated immunoassays in a single experiment, Sally delivering 96 automated immunoassays in a single experiment, both of which are based on a very robust capillary electrophoresis technology, which I will describe to you in more detail

The Simple Western really delivers three main benefits, separation by molecular weight, target ID and specificity, using any primary antibody of your choice, and robust and precise quantitation

So, as you can see in the separation diagram on the left, we generated an electropherogram, which can be fitted by either area under the curve or P-kite [sp], allowing very precise and accurate quantitation

The unique gel-free, blot-free, and hands-free format of the Simple Western also provides unmatched throughput, FTE efficiency, and data standardization, all of which I'll cover in this presentation

The Simple Western platform provides new capabilities. The data that you're seeing here is 96 samples run in a single experiment

And the really amazing thing about this data isn't the 96 samples. It's the fact that you can use multiple detection antibodies in a single experiment. You can actually use up to 96 different primary antibodies in one single experiment, something that is impossible to do with any traditional method today

You can also ask up to 16 immunoassay questions from one small five-microliter sample. The Simple Western platform actually only requires nanoliters of material inside the capillary. So, by starting with five microliters in a plate, you can ask a lot of questions

As I did say a couple of times, you can with the Sally instrument analyze up to 96 samples at a time. And that also allows you to efficiently optimize your assay in a single experiment

You can eight capillaries for one parameter, eight capillaries for another parameter, and so on, and in one simple experiment arrive at a fully optimized assay

The Simple Western is more than just an instrument. It's a complete solution. ProteinSimple offers kits and software that go along with each instrument, allowing plug-and-play assays

And if you think about it, Simple Western is really the future. As you look today, you would prepare your samples and put them into a microplate

You'd then put them into the instrument and press "start" and then go about your day

The Simple Western takes care of the rest by delivering results automatically

Once you put the samples into the instrument, there's no further intervention required

So, I'd like to describe next how the Simple Western works. With the samples now in the instrument, the instrument can in a fully automated way load the separation matrix into the capillary, draw the sample into the capillary, separate the sample in matrix automatically, immobilize using a proprietary UV technology, and then immunoprobe by drawing both the primary and the secondary antibodies into the capillary

It then automatically quantitates the data. And this process is fully automated. Unlike a traditional Western, there is no gel or apparatus preparation, lengthy and cumbersome hydrophobic transfer to a membrane, manual incubation or wash steps, and certainly no standing in a dark room developing or wasting film

Just to illustrate a little bit better how this works, I'd like to show you some of the internal components of the two instruments

So, what you're seeing here is the front of the Simon instrument. And at the bottom right-hand corner of the screen, you can see the capillary cartridge that contains 12 capillaries

Those capillaries are moved between the assay plate compartment, which is in the very top left, to the incubation tray and separation block, which you can see right in the middle. And all of the steps are automated so that the user, again, has no intervention in this process

The Sally instrument, because of its throughput, has two slots for capillaries. And you can see in each slot are significantly more capillaries than in the Simon instrument. So, in total, this instrument can hold up to 96 capillaries

And it also holds some reservoirs for wash buffer and running buffer, again, so that the process inside the instrument can be fully automated

The Sally also has several interior compartments or trays that designate where different steps take place. So, there's a separation detection tray, incubation trays, and so on so that the instrument can automate the different steps at the appropriate temperature and condition

This results for the user various views of the data. On the left is a capillary image view. And actually, inside the software, this is a movie that one can watch the separation take place in sort of an accelerated timeframe

The middle is the electropherogram view, which is where the raw data really exists. And it's also where the quantitation of the protein in the capillary can take place

And then the software can generate a virtual Western blot view called the lane view, for those used to seeing data in this format

I'd like to switch a little bit from the instruments now and talk about the data that can actually be generated with the Simple Western

The Simple Western can deliver separation of proteins based on size from 15 to 150 kiloDaltons. And as you can see in this dataset, using different primary antibodies, you can separate proteins that run at different molecular weights

The molecular weight as assigned by the Simple Western is very similar to the molecular weight that you would find, either through the sequence data or by traditional FDS page Western blot analysis

And once proteins are separated, they can easily be identified again, using the primary antibody of your choice

In this particular example, we were looking at ofusnucleon [sp] in both wild-type mouse brain and a human cell line one

And you can see that you can easily compare the amount of those proteins present in each of those cell lines by using a primary antibody

And this can be done for any protein. That includes tagged proteins, like His-tag proteins, FLAG-tag proteins, etc

If you have the primary antibody for it, you can identify your protein in the Simple Western

The ultimate culmination of these two capabilities, size separation and ID using primary antibodies, allows one to do pathway profiling with the Sally

But, it's not pathway profiling like you've ever seen before because, again, you can use multiple primary antibodies in a single experiment

So, let me just describe the data that you're seeing now

This is profiling of the NF-kappaB pathway with eight different primary antibodies in stimulated versus unstimulated cells in whole cell versus nuclear extract in replicate

All of this is done in a single experiment. And each sample was only a five-microliter sample. So, for every five-microliter sample, we took eight subsamples and used a primary antibody for each of those subsamples. We essentially asked 16 questions

The ability to separate by size and to identify those peaks based on using a primary antibody can only be complemented in a meaningful way through quantitation. And that's what I'm describing to you now

The Simple Western can reproducibly and quantitatively analyze samples in the capillary. And the electopherogram view is how we accomplish that

As you can see in this slide, it's connection 43. You can precisely determine how much is in each capillary of that particular protein using the appropriate primary antibody

You can also very easily get accurate mass per mil or mass-for-mass quantitation by simply running a dilution curve of your standard, generating a standard curve and extrapolating the unknown values against the standard curve

And as you can see, across replicates and across your standard curves, you can achieve very precise data. In this example of quantitation of N-pap-C1 [sp], all components of the standard curve had a precision of 6 percent or less

I'd like to switch gears just to talk about throughput with the Simple Western. Throughput with the Simple Western is unlike anything you've ever seen before for a couple of different reasons, the primary one being the data just in front of you

In a single experiment, you can achieve up to 96 answers at once and with precision that is unmatched by any traditional method today

But, throughput isn't just that simple. It's not just how many samples can you run. It's really how much more can you do with the time that you have. And I'd like to talk about that next

In the data that you're seeing from Alice Fan from Stanford University, Alice screened 100 patient samples using Sally. And the samples were a mixture of tumor-containing samples and non-tumor or standard samples

And each sample was run five times. You can see, although the error bars are very small, there are error bars on the top of each one of these bars

But, looking at ERG1 and ERG2, she was able to look at the differences between the patients that expressed tumor and those that didn't. And she can do that in about a three-day period

So, essentially, she ran 500 samples in a three-day period

Just to show you some numbers, it really works out as shown on this slide. So, here's an example of analyzing 312 samples

Three hundred and twelve samples is really a comparison to running 12 26-lane traditional Western gels versus running four runs of 78 capillaries on the Sally. And of course, Sally could run up to 96. But, for the purposes of the comparison, we're going to limit it to 78

Running 12 26-lane SVS-pace [sp] gels in the traditional format would take at least a full week, maybe slightly more than a full week, a day and a half for separation. Depending on whether you have an automated blotting station, it could be half a day to as many as two days for the transfer step, another day to expose film, and then two days to analyze all of this manual data

The Sally can do this in less than half of that time. Because the process is automated, really, it's two and a half days of instrument running time and just about two hours or so of data analysis time

So, just on time alone, Sally can deliver two to four times more throughput than traditional methods

But, where it really shines is that Sally can deliver organizational savings on time. That traditional Western method that took a whole week to complete and 32 hours of real hands-on time now only takes two and a half days using Sally but only requires six hours of hands-on time

That means that Sally delivers 26 hours every week available for you to do something more meaningful or more important to your organization

And on top of that, for the first time, in a platform that delivers size-based separation, target ID specificity, and quantitation, you really have a standardized approach to compared data site to site, lab to lab, and person to person

So, just to summarize, I hope I've shown you today that the Simple Western really delivers more, delivers size-based separation. It delivers target ID and specificity using primary antibodies of your choice, real quantitation, very good reproducibility, efficiency, and throughput, all in a single package

If you compare that to the other tools that you probably have in your laboratory today, none of those tools can deliver all of those things by themselves

So, I'd like to say, "Thank you for joining me today." I hope you've enjoyed this introduction to the Simple Western platform. And I hope you've seen that this gel-free, blot-free, hands-free platform really can deliver more