How to Control Your Apoptosis, Autophagy, and Mitophagy Western Blots

Have you ever spent time treating cells, preparing cell extracts, and performing a western blot only to be disappointed when you don’t see the expected signal or change in protein expression? That inevitably leads to the next question–did something go wrong with the experiment? Using control cell extracts as positive and negative controls in your immunoblots can tell you if there is an issue and, if so, help identify the problem.

Did you see no change in protein expression after treating the sample, but you did see a change in signal with the control cell extracts? Then the issue doesn’t lie with your antibody, and you may want to take a closer look at your sample preparation.

Control cell extracts are especially useful if you’re investigating cell death-related events for the following reasons:

• Signal Induction: Cell death pathways are extremely nuanced, making it sometimes difficult to induce the desired cell death pathway in your sample. Different treatments can lead to the activation of different signaling cascades.
• Sample Preparation: It takes experience and the right protocol to know what sample preparation method is optimal.
• Sample Collection: Cell death-related targets often undergo transient post-translational modifications (PTM), like a cleavage event, so you could be preparing extracts at the wrong time point.

Cell Signaling Technology offers a few different control cell extracts, each of which consists of a negative and positive control that can be used for your cell death western blot experiments. The negative control contains either no or lower endogenous levels of the protein or PTM compared to the positive control, which contains higher levels of the induced protein/modification of interest. As a bonus, most of the compounds used by CST to induce cell death in the control cell extracts, like etoposide, chloroquine, and thapsigargin, are also available for you to treat your own cells with if you want to minimize experimental variables as much as possible.

This post explores the best control cell extracts to use as positive or negative controls in your western blots when studying apoptosis, autophagy, and mitophagy.

Apoptosis is a type of programmed cell death that is mediated through caspase cleavage events as part of the cell death pathway. It is characterized by cell membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA decay. Defects in apoptosis are implicated in cancer, neurodegeneration, cardiovascular disorders, and autoimmune diseases.¹ Apoptosis can be chemically induced in cells using either etoposide or cytochrome c.^2-3

CST offers two control cell extracts for detecting apoptosis via western blot:

• Jurkat Apoptosis Cell Extracts (etoposide) #2043
• Caspase-3 Control Cell Extracts #9663

The positive controls for Jurkat Apoptosis Cell Extracts (etoposide) #2043 and Caspase-3 Control Cell Extracts #9663 are produced from total Jurkat cells treated with 25 µM etoposide for 5 hours or the cytoplasmic fraction of Jurkat cells treated with cytochrome c, respectively. Both can be used as control cell extracts for your apoptosis western blots.

Figure 1. Representative data from Caspase-3 Control Cell Extracts #9663 new lot testing. Western blot analysis is shown for Caspase-9 Antibody #9502 (top) and Caspase-3 (D3R6Y) Rabbit mAb #14220 (bottom). As expected, cleavage of caspase-3 and -9 are induced upon cytochrome c treatment (+). Full length forms of both proteins are seen in untreated extracts (-). Lot-to-lot signal is maintained. 

Below are some of the targets for which you can use Jurkat Apoptosis Cell Extracts (etoposide) #2403 and the Caspase-Control Extracts #9663 as controls when running a western blot to examine apoptosis.

The CHOP Control Cell Extracts #33263 positive control is total cell extracts from C2C12 cells treated with 300 nM thapsigargin for 2 hours and can be used to investigate CHOP expression and other mitophagy targets in your western blot.