Read our blog on how new methods increase precision in protein visualization
Here we describe some of the great solutions that are coming out of recent advances using CRISPR CAS technology that give more precise and physiological results for protein visualization. In our previous blog (see link at end of article), we discussed the some of the difficulties with traditional methods for protein tracking and localization. One of the main causes of variability and wasted resources is the lack of standards for antibody quality.
To be useful, an antibody must:
Have a high signal to noise ratio
Be validated for the assay at hand
Efforts to reduced non-specific antibodies in both industry and academia
From an industry standpoint, numerous organizations and commercial suppliers have created (or are creating) programs to ensure that the above criteria are met for each new antibody brought to market. The Human Protein Atlas is one of these groups. (Listen to our interview with Emma Lundberg of the HPA here.) Horizon Discovery has also contributed to raising the awareness of the importance of anitbody quality, and have partnered with Abcam, leaders in antibody characterization, to provide gene-edited cell lines for antibody validation. Academia, as one of the biggest users of antibodies, has also been vocal in raising concerns over antibody quality. Dr. David Rimm, Professor of Pathology and of Medicine began promoting a specific algorithm for validation in 2010 with publication of a paper in Biotechniques, and numerous other examples can be found.
From the technical side of things, there are general protocols that can be followed to test any new antibody. The most rigorous method is a two-step process that takes advantage of current gene editing technology. One option for the first step is to test an antibody on cell lines that have undergone knockdown/silencing through siRNA or another form of RNAi. An antibody performing as intended should provide minimal to no signal in these cells, and any signal due to protein remaining after knockdown should of course be localized to the expected cellular compartment (assuming that is known). To validate knockdown, one can use an already validated antibody on a western blot and/or RT-PCR to check mRNA levels.
However, a more thorough option is to use gene-edited cells where the gene of interest has been removed completely. Knockout cells and tissues have been held up as an ideal control for antibody testing for many years. In 2003, Saper and Sawchenko stated,
“The gold standard for deciding this issue is to stain tissue with and without the antigen of interest. This can be accomplished by staining tissue from a wild-type mouse and one in which the antigen of interest has been eliminated by transgenic engineering.”
Using the “gold standard” is more effective today with the introduction of the CRISPR-Cas9 system. Rather than hoping knockdown has taken place, CRISPR-Cas9 removes the target gene completely. Then, confirmation should be carried out through RT-PCR to ensure the absence of the relevant mRNA. The resulting cell lines represent a true negative control.
The second step is to determine whether signal from the antibody represents true labelling of the protein of interest. For a protein such as actin, this is relatively easy due to the distinct nature of F-actin filaments. However, It is best not to rely on a known localization pattern, no matter how distinct. Furthermore, doing so is obviously impossible when looking at a novel protein or a known protein in a novel context. Researchers need a point of reference for determining the quality of labelling provided by a new antibody. Here again, gene editing is a valuable tool.
Traditionally, fluorescently-tagged (FP)-proteins ave been introduced into cells through classical molecular biology techniques such as transient transfection. If the expression vector contains a GFP-tagged gene of interest, the researcher would simply use an mCherry secondary antibody to detect the primary antibody being tested. Colocalization of the two signals plus the absence of either green or red signal exclusively, would indicate that the antibody is indeed labelling only the protein of interest.
As mentioned above, however, overexpression systems are prone to problems such as highly variable expression levels between cells and mislocalized protein. A better option is to use CRISPR-Cas9 to insert a cassette containing an FP-tagged version of the protein of interest at the native loci. Doing so puts the target protein under control of its native promoter, and its expression will be consistent with that occurring in a non-tagged cell. From there, the researcher can compare localization of the antibody signal with signal from the tagged protein.
Emma Lundberg and colleagues recently published a detailed protocol for validating antibodies with endogenous expression of tagged proteins. Lundberg is currently using this process in her work with the Human Protein Atlas to test the hundreds of antibodies in that group’s catalogue.
Horizon Discovery and Antibody Validation
Horizon Discovery is deeply invested in the push for better antibody validation. Our partnership with Abcam is one clear step to ensure that reagents on the market are providing accurate results for researchers to build from. However, our work in this arena goes beyond the partnership and is available to scientists around the world.
Horizon offers a large catalogue of knockout cell lines that have been created through CRISPR-Cas9 engineering in the HAP1 cell line. With complete loss of gene function, these HAP1 cells can be used for the first step in antibody validation, looking for signal in a knockout context.Gene-edited HAP1 cell lines are also available for the second stage of antibody validation, comparing antibody signal with that from an endogenously-tagged protein. Horizon Discovery works with Promega Corporation to develop custom reporter cell lines expressing a TurboGFP-tagged version of any protein of interest. Researchers can then use these lines for comparative localization studies when testing new antibodies. Furthermore, HAP1 cells expressing a tagged protein can be used for live-cell imaging assays to track and localize the relevant molecule, adding another dimension to the static data acquired from fixed samples.
Emma Lundberg of the Human Protein Atlas pointed to this benefit and the complementary nature of antibodies and tagged proteins in a recent conversation with Horizon :
“Tagged proteins and antibodies are very complimentary and if you work in cell lines, tagged proteins are great because you can study dynamic processes in the living cell. You cannot do this with antibodies, but when it comes to studying human tissues, you cannot easily tag proteins in them so you’re stuck with antibodies.” You can hear more from the interveiw with Dr Lackner from our blog post here.
The Value of HAP1 Cells
HAP1 cells are particularly well-suited for knockout experiments or for inserting cassettes encoding a tagged protein. As the name suggests, HAP1 cells are haploid, so only one loci needs to be altered to obtain the desired effect. Daniel Lackner, Team Leader at Horizon Discovery, explained: “The great advantage is that you only have one copy of the whole genome. That allows us in a very precise way to generate mutations. We can easily genotype and find out where its mutation is, to see if we have done what we wanted to do. In a diploid cell you might have a scenario where you generate a mutation and it's only in one allele in one chromosome, whereas the second one is not harmed and you still have a functional protein. In a haploid cell line like the HAP1s, as soon as we generate a mutation we have, in most cases, destroyed the gene.”
The same is true for the FP-tagged cell lines:
“The great advantage here is the fact that once you have integrated the tag and you have generated your tagged gene, 100% of the protein in the cell will have the tag. There's not a second allele that does not contain the tag.”
Put simply, Horizon has developed cell lines that dramatically simplify the process of testing reagents used for evaluating protein expression. HAP1 cells provide a near-binary readout because of their haploid nature, something that is quite uncommon in the heterogeneous world of cell culture. Furthermore, Lackner and the Horizon team have modified the CRISPR-Cas9 system so that long, specific guide-RNAs are no longer required for editing protocols. Instead, a somewhat generic cassette containing the tagged gene is inserted using non-homologous end joining. This cuts down on the time and cost required to create a new cell line, savings that are passed on to the researcher. You can hear more from the interveiw with Dr Lackner from our blog post here.
There are many ideas for producing better antibodies. Some, such as using sequencing to build recombinant antibodies, utilize bioinformatics approaches that should be consistent and high-quality. Regardless of the production technique, however, all antibodies must be fully validated prior to commercial distribution. Horizon Discovery is committed to working with the scientific community to ensure that each antibody used in published work targets its intended protein - and only that specific protein. Our HAP1 knockout cells, along with our TurboGFP reporter lines, represent a powerful contribution to the process of antibody validation, as well as to associated assays such as live-cell imaging.
To see how Horizon's cell lines are being used for protein visualisation, visit our website:
For questions or comments regarding any of the Reporter cell lines from Horizon Discovery (including NanoLuc and HaloTag versions), contact us here.
Alternatively, see our previous blog "Spying on proteins...", where we summarized a growing body of work describing the issue of poorly characterized antibodies in basic research.