Cell lysates are essential experimental tools which laboratories use for a plethora of applications in drug development, translational research, and even synthetic biology. As reagents, cell lysates are inexpensive, rarely interfere with detection assays, and are comprised of a set of components which can be defined in detail if experimentally necessary.
While there are many roles which lysates may fill, lysates are most commonly used for:
- Evaluating antibodies
- Measuring enzymatic activity
- Gel electrophoresis
- Ligand binding studies
- Western blotting
Each of these applications requires a different experimental protocol and lysate titration. But, as most lysates are pre-homogenized and formulated with SDS buffer, the same vial of lysate can be used in many different applications once the appropriate experimental preparations are complete. By learning a practical routine for utilizing lysates appropriately in these contexts, researchers everywhere will be able to unlock the powerful and versatile capabilities of lysates.
Assessing Antibody Activity Using a Lysate
Lysates have a long history of use as workhorse tools for assessing antibody activity in the context of ELISA or another immunosorbent assay. To assess antibody effectiveness using lysates via ELISA, first coat an ELISA plate with the antibody samples of interest, making sure to leave at least one well uncoated as a negative control. To guarantee that at least a portion of the plate will be within the range of detection after development, it is often useful to make a matrix of dilutions of the antibody under investigation when coating the plate.
Next, select the appropriate lysate and make a couple of dilutions using the appropriate ELISA incubation buffer. Be sure to use a lysate which could plausibly contain antigens that the antibody of interest could bind to under the conditions of the incubation buffer in the ELISA plate. Similarly, don’t forget to assign a positive control well and an additional negative control well to verify that the plate is developed properly.
Then, begin to develop the ELISA plate while maintaining a uniform total volume in each of the plate’s wells during each phase of the process. Before adding the developing substrate to the ELISA plate, wash the plate carefully. While commercial ELISA kits often include instructions for plate washing, washing once or twice more than suggested is advisable when running ELISA using lysates for the purpose of antibody evaluation. Finally, add the developing substrate and monitor the change in color of the plate.
When using tissue lysates for antibody evaluation, over-development of ELISA wells should be a primary concern. Stop the reaction as soon as there is a clearly visible gradient of color between the different dilutions of the antibody of interest or the different dilutions of the lysate. Then, use a spectrophotometer or an ELISA plate reader to measure the hue of the wells in the plate. By comparing the hues on the plate, the concentration of the antibody, and the concentration of the antigens in the lysate, it is subsequently possible to calculate the affinity that the antibody has for the components of the lysate.
Using Lysates In Electrophoresis
Lysates are also useful during gel electrophoresis of proteins because they can be used to establish a ladder with known sizes relative to unknown components of interest. To use lysates during electrophoresis, ensure that the lysate is suspended or diluted at the same concentration into the same buffer solution as the other samples which will be run on the gel. In most cases, this means that ensuring the lysate is in the SDS buffer. Next, establish the lysate’s lane next to any other reference ladders which will be used in the experiment, taking care to identify the proteins within the lysate which will end up nearby benchmarks established by the other reference ladders after the gel has been run. Then, run the gel, making sure to stop the electrical current well before the benchmark ladders are compressed beyond measurement.
Interpreting a lysate used during electrophoresis is very similar to interpreting any other electrophoresis result. Proteins within the lysate will form bands along the gel depending on their molecular weight. However, as lysates have a plethora of different components, lysates which are too concentrated will appear on the gel as a smear. Thus, it is important to dilute lysates before running them on a gel if the goal is to compare the known components of the lysate to those of another sample.
Picking The Right Lysate
Before a laboratory can use cell lysates, it’s necessary to pick the correct lysate for the job. In general, the correct lysate is the lysate which is pertinent to the desired downstream application. For example, if an experiment sought to investigate the bulk size of tumor antigens relative to known cellular proteins, the appropriate lysate would be the lysate of the type of tumor cell which constitutes the tumor.
Because it may be difficult to access the exact type of cell that is the downstream target for some assays, it may be necessary to use a lysate of a closely related cell or a commercial tumor cell lysate instead. Consequently, for pathology-specific work, pairing clinically-defined cell lysates from healthy tissue and a lysate from clinically-defined tumors in the same tissue can be highly effective.
The intended experimental apparatus is also a relevant concern for picking lysates. Lysates which are formulated without a reducing agent are necessary for experiments where protein binding is of principal importance. This means that for lysates destined for use in ELISA and ligand binding studies, only reducing agent-free lysates will work.
Controlling For Lysate Unknowns
There are many experimental applications which may require knowing the complete antigen complement of a lysate. In these cases, there are several approaches which can shed light on the antigen makeup of a given lysate. The most obvious and the most expensive approach to defining the antigen makeup of a lysate is mass spectrometry. However, for most applications, Western blotting the poly-filtered, centrifuged, or ultracentrifugated fractions of the lysate will likely be sufficient to gain an understanding of the abundance of the relevant antigens and their molecular masses. Alternatively, using a mixture of antibodies and analyzing the lysate via flow cytometry will also shed light on the disposition of a lysate’s antigens of interest.
Lysate Titration Is Critical
Having the right lysate and understanding its components are only the first steps toward operationalizing lysates in an experimental setup. Titrating the volume of lysate is largely a process of trial and error, but a quick titration experiment for the intended application doesn’t need to be difficult. For an experimental application using ELISA, titrating lysates could be as easy as developing each row of an ELISA plate with a different dilution of the lysate. Subsequently, if a well of the plate is over or under-developed, the concentration of the lysate is too low or too high.
Similarly, if the lysate’s detection assay appears to be noisier than desired, reducing the concentration of the lysate and washing the relevant plates to remove excess lysate can be helpful. In contrast, in some experimental setups, removing the lysate before running a detection assay or functional assay may be unnecessary, making the initial titration process significantly easier.
Pushing Lysates To The Limit With Healthy Tissue/Tumor Pairs
While cell lysates are highly effective, they are also exceptionally versatile. Though it’s unlikely that cell lysates will be actively developed to increase their efficacy at performing any single one of their myriad applications, their low cost and their ease of use will continue to make lysates critical experimental tools for years to come. Sourcing lysates for experimental applications doesn’t need to be difficult, and working with a reputable lysate supplier can make finding the right lysates even easier.
Cell Sciences is introducing a human tumor lysate collection from a diverse range of cancer pathologies from over 20 organs, each of which includes a matching adjacent normal tissue lysate. Using matching lysates allows for easy setup of comparison studies and accurate biomarker identification, meaning that these new lysates have the potential to streamline the research process. Please inquire regarding lysates from specific pathologies at email@example.com.