Video: Transferring Your Traditional Western Blot to Wes

Learn How To Transfer Your Traditional Western Blot

Hello, I am Susan Harrison Uy from ProteinSimple, and today I'm going to walk you through the process of transferring your traditional western blot to our ;Simple Western platform Wes giving you some tips for optimization and implementation along the way.

Traditional western blotting is a commonly used technique for protein analysis, but it presents some technical challenges that may impact experimental reproducibility

West is a capillary based immunoassay system that eliminates many of the challenges that you may face with traditional Western blotting.

All steps are automated eliminating some of the risk of human error that you may encounter with traditional Western blotting.

That means that everything from protein separation, immunoprobing, detection, and data analysis occurs within the system

Wes also gets you down to picogram level sensitivity with just three microliters of starting material enabling you to get more out of your precious sample.

Wes can run 25 samples in 3 hours giving you quantitative, size-based data including total protein.

Now, I'll walk you through what happens inside the Wes instrument.

Western Blots Inside The Wes Instrument

All of your reagents, sample, antibodies, and detection components are loaded from a specially designed plate.

Wes begins by aspirating the separation matrix and then the stacking matrix into the capillaries.

Next, your sample is loaded and then the capillaries are lowered into the running buffer.

Voltage is applied which enables separation of your sample by molecular weight.

Then, the immunoprobing process begins first with incubation with your primary antibody, then your secondary HRP conjugate, and finally the chemiluminescent detection reagents.

The chemiluminescent reaction is imaged in a series of images by a CCD camera over time.

3 hours later, you'll get quantitative size-based data.

Now, I'm going to walk you through a workflow optimization using an example of the detection of AKT.

Optimization Workflow Setup

The first step in setting up your Wes assay is to choose the antibody that you want to use.

You can either use antibodies that you already have from your traditional western blotting process or you can search the Simple Western antibody database - an open repository of thousands of antibodies with proven results for Simple Western assays.

The next thing you need to do is to identify the model system that you're going to use to optimize your assay.

The systems should contain a positive and a negative control in order to enable you to detect activation or expression of your target of interest.

In our assay example, we're using a positive control of phosphorylated AKT cell extract and the negative control of nonphosphorylated AKT cell extract.

Step 1: Initial Antibody Screen

The first step in optimizing your Wes assay is an initial antibody screen.

In this step, we're trying to find the right antibody in terms of signal intensity and specificity.

In this example here, we tested two total AKT antibodies and three phosphorylated AKT antibodies.

In order to enable detection of phosphorylated and total AKT in the same lysate, we treated the cell lysate first with Calyculin A.

Both total AKT antibodies were able to detect AKT, but one had higher signal, so we decided to move forward with that antibody.

Of the three phosphorylated AKT antibodies, one resulted in nonspecific bands, so we eliminated that from our process.

The remaining two antibodies were also screened against negative control tissue, and the best-performing antibody was chosen to move forward in our assay.

Step 2: Determine Lysate Concentration

Next, we ran a lysate dilution in order to determine the optimal lysate concentration for the detection of our protein of interest.

Linear regression analysis identified that this chemiluminescence signal continued to increase linearly up to 0.1 mix per mil and then a plateaued for both antibodies.

We therefore chose to use a concentration of 0.1 mix per mil to enable detection of our protein within the linear range of the lysate.

Step 3: Determine Antibody Concentration

The final step in our assay optimization is to run an antibody dilution series to determine the optimal antibody concentration to use.

In this case, we tested a series of antibody dilutions from 1/25 to 1/800.

To ensure reliable quantitative detection of our protein of interest, we need to choose an antibody dilution that enables detection of our protein of interest below the level of saturation.

In this case, we chose a 1/50 dilution for both antibodies.

With all this information gathered, you're now ready to run your optimize assay.

Run Optimize Assay

In this case, we worked with a 0.1 mix per mil lysate concentration and a 1/50 antibody dilution for both antibodies.

To test our system, we treated the cell lysate with Calyculin A, and we were able to observe a two-hundred-and-eighty-fold increase in phosphorylated AKT expression in our Jurkat Cell Lysate.

In order to quantify your protein expression, you'll need to normalize your protein expression.

Many people use housekeeping genes in order to enable this quantification.

However, total protein expression more accurately reflects endogenous protein expression resulting in better quality data.

With Wes, you can use the total protein detection module with any Simple Western immunoassay module to enable quantification of total protein.

In this example, we detected total protein expression shown in even number lanes and AKT expression shown in odd number lanes using the same sample.

Multiplex Data Points

Multiplexing gives you the flexibility to increase the number of data points that you can collect per sample.

In this example, we used a Rabbit Anti-ERK Antibody to detect ERK1/2 expression and a Mouse Anti-STAT 3 Antibody to detect STAT 3 expression.

As long as your proteins of interest are of different molecular weights and do not comigrate, you can even use antibodies derived from the same host.

Simple Western with Wes lets you separate and analyze proteins by size from two to four hundred and forty kilodaltons.

With just three microliters of starting material, Wes can run up to 25 samples in three hours giving you quantitative size-based data including total protein.

For more information on how to implement Wes in your lab, please check out our app note resources. Thank you.

Learn more about Wes by ProteinSimple, a Bio-Techne brand, by visiting the Wes instrument page.