Converting a Popular Puromycin Antibody to Recombinant Production

The laboratory of Scot R. Kimball at Penn State College of Medicine developed an anti-puromycin antibody that enabled non-radioactive measurement of global protein synthesis. Since its initial development in 2013, scientists around the world have used the antibody to study a variety of physiological functions, as well as conditions such as cancer and Alzheimer’s disease.

The puromycin antibody was originally developed in the mouse IgG1 format and generated from a hybridoma cell line. To ensure the longevity of this useful research tool, Absolute Antibody sequenced and recombinantly produced the antibody. We also engineered it into additional species and isotypes to open up new experimental possibilities. Data show the recombinant puromycin antibodies in both mouse and rabbit formats perform as well as the widely used hybridoma-derived antibodies, while offering options for reducing background signals.

Developing the Antibody for Disuse Atrophy Research

Research in the Kimball lab focuses on disuse atrophy, or the loss of muscle mass from lack of physical activity. In particular, they sought to determine why protein synthesis decreases in inactive skeletal muscle. To answer this question, the lab needed a simple method of measuring global protein synthesis, or mRNA translation.

Traditional pulse-chase techniques relied on radioactive labeling, which could be cumbersome, expensive and potentially dangerous. The Kimball lab developed an easier procedure, using an antibody to puromycin, that was just as effective without the need for radioactively labelled agents. Puromycin incorporates into newly synthesized peptides, and the puromycin antibody allowed for direct evaluation of translation using standard immunochemical methods such as Western blot and ELISA.

Accelerating Adoption of the Puromycin Antibody

As peer-reviewed research was published using the puromycin antibody to measure protein synthesis, more labs wanted access to the antibody for their own studies. To facilitate sharing of the antibody, the Kimball lab partnered with our sister company Kerafast, whose mission is to make unique laboratory-made research tools easily accessible to the global scientific community.

The puromycin antibody was added to the Kerafast catalog, where it could be purchased by researchers worldwide without undergoing the traditional and time-consuming Material Transfer Agreement (MTA) process. As Kerafast began to market the antibody, demand quickly increased. To date, Kerafast has helped fulfill more than 1,000 requests for the antibody, sending shipments to scientists in 35 countries across five continents.

“As the investigator providing materials, the Kerafast program saves a lot of time and a lot of my effort,” Dr. Kimball said. “It’s definitely a much quicker and much easier process than the traditional way of exchanging materials.”

Antibody Sequencing, Engineering and Expression

After the merger of Kerafast and Absolute Antibody, which have now come together under the parent company Absolute Biotech, researchers whose reagents were available via the Kerafast catalog had easy access to Absolute Antibody’s recombinant antibody technology. The puromycin antibody was therefore sequenced using our NGS hybridoma sequencing approach, enabling both antibody engineering and recombinant production.

“There are a couple of reasons why I was interested in producing a recombinant anti-puromycin antibody,” Dr. Kimball said.  “First, and probably most important, is that we don’t have to worry about ‘losing’ the hybridoma cells that we currently use to produce the monoclonal antibody. Over the years we have had hybridoma cell lines stop making antibody for no apparent reason and we’ve had to ‘start over’.”

He continued, “Second, the species and isotype can be changed. For example, the monoclonal anti-puromycin antibody was raised in mice, and if you use it to detect puromycin incorporated into proteins in mouse tissue, e.g., skeletal muscle, you have to use an anti-mouse IgG secondary antibody that can recognize endogenous mouse IgG antibodies and give ‘background’ bands on Western blots that need to be accounted for during quantitation. A different species IgG (or an IgM) would avoid that problem.”

The puromycin antibody is now available in recombinant engineered formats in both the Kerafast and Absolute Antibody catalogs. Available formats include the original mouse IgG1, rabbit IgG, mouse IgM, mouse Fab fragment, human IgG1, and human IgG1 with an Fc silencing mutation that further reduces non-specific background in staining methods. In addition, the antibodies can be ordered with custom conjugations, such as a fluorescent or biotin tag, to remove the need for a secondary antibody in Western blot or immunofluorescence applications. Other engineered formats are always available upon request with Absolute Antibody’s custom services.

Data generated by the Kimball lab demonstrates the new recombinant antibodies perform just as well as the original hybridoma-derived antibodies, if not better (see figures below). Figure 1 compares the original antibody to the recombinant anti-puromycin antibody in mouse IgG1 format. Figure 2 also looks at the recombinant antibody in rabbit IgG format. The rabbit format provided a cleaner gel as there was no background interference from endogenous antibodies in mice. Dr. Kimball therefore recommends either the recombinant mouse or rabbit versions of the antibody for assessing puromycin incorporation in cultured cells, but for mouse in vivo studies, the rabbit format should be used.

Researchers worldwide can now use the recombinant anti-puromycin antibodies to measure global protein synthesis as confidently as they would use the original antibodies – while benefiting from the many advantages recombinant production offers. With the recombinant anti-puromycin antibodies, researchers can be assured of the long-term batch-to-batch reproducibility of their research tools, as well as custom engineer the antibodies into any species, isotype or format to further their experiments.

Try the Recombinant Puromycin Antibody – Save 30% On Your First Order!

Are you interested in trying a recombinant version of the puromycin antibody for your research? We’re offering a 30% discount on a single order of a recombinant puromycin antibody! Use the discount code 3RH30 at checkout, or contact us for the discounted quote. The code applies to one standard sized vial and is good through the end of 2023.

If you’re interested in studying translation, Absolute Antibody, Kerafast, and their sister brands carry a variety of antibodies targeting translation factors, including:

If you have any questions, or are interested in converting your own antibodies to recombinant production, please don’t hesitate to get in touch. Our team has converted thousands of antibodies to recombinant production, both as custom antibody services for clients worldwide, and to build our catalog of engineered recombinant antibodies. We look forward to discussing your project!

Figure 1. The left Western blot was probed with the original monoclonal anti-puromycin antibody, while the right Western blot was probed with the recombinant anti-puromycin antibody in mouse IgG1 format, both at 1:1,000 dilution. The secondary antibody was used at the same dilution for both sides and they were both exposed for 40 seconds.
In both Western blots, the first two samples were from skeletal muscle of mice, where the first lane is muscle from the control hindlimb and the other is from a hindlimb that had been immobilized with a cast for three days. The other three lanes are from HEK393T cells: the first lane is from cells incubated in complete medium, the middle lane is from cells incubated for 2 hours in medium lacking glucose and serum, and the last lane is cells incubated for 1.5 hours without glucose or serum and then glucose and serum were returned during the last 30 min. As expected, puromycin incorporation was lower in the immobilized hindlimb compared to the contralateral control hindlimb, and also in cells deprived of glucose and serum compared to cells in complete medium. Resupplementation partially restored incorporation.

 

 

Figure 2. This figure compares the Western blots of the two mouse IgG1 antibodies (hybridoma-derived monoclonal and recombinant) with the recombinant rabbit IgG antibody (right blot). The effectiveness of the rabbit antibody is comparable to that of the mouse antibodies, while the rabbit antibody also prevents the non-specific bands of interference in the two mouse antibody blots, likely caused by secondary antibodies picking up mouse proteins. The arrows denote the heavy and light chains of mouse IgG, which are completely absent in the rabbit antibody blot.

Latest News

Upcoming Events

Please join us at the following conferences and events. Stop by our booth, or get in touch to arrange a meeting.

See All Dates

Next Event:

ADLM – Booth #1245

28th July - 1st August 2024

Chicago, Illinois

Chimeric vs. Humanized Antibodies

Read more

Antibody Sequencing and High Throughput Production for Hybridoma Libraries

Read more