Purification of Mitochondrial Proteins HSP60 and ATP Synthase from Ascidian Eggs: Implications for Antibody Specificity

Abstract

Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates). The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.

Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria‐rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo

“…; Chenevert et al . ). Interestingly, exomitochondrial F 1 ‐ATPase subunits have been proven to bind to some cytoskeletal filaments and myoplasmin‐C1 (Nishikata & Wada ) and are thought to form a novel complex without F 0 subunits.…”

“…Previously, we revealed that the aand b-subunits of F 1 -ATPase are exomitochondrially localized to the cortical region of the myoplasm in the ascidian egg (Ishii et al 2012;Chenevert et al 2013). Interestingly, exomitochondrial F 1 -ATPase subunits have been proven to bind to some cytoskeletal filaments and myoplasmin-C1 (Nishikata & Wada 1996) and are thought to form a novel complex without F 0 subunits.…”

“…; Chenevert et al . ). These subunits were unexpectedly found to exist in the cytosol and are suggested to play unknown roles other than mitochondrial respiration (Ishii et al .…”

“…These maternal factors are important for the establishment of the anteroposterior axis, unequal cleavage, cell fate determination, and a number of cellular processes. In addition, we previously revealed that the aand b-subunits of F 0 F 1 -AT-Pase, which forms complex V of the mitochondrial respiratory chain, are localized to the cortical region of the myoplasm (Ishii et al 2012;Chenevert et al 2013). These subunits were unexpectedly found to exist in the cytosol and are suggested to play unknown roles other than mitochondrial respiration (Ishii et al 2012).…”

The Spindle Assembly Checkpoint Functions during Early Development in Non-Chordate Embryos

“…Membranes were incubated in 2% milk with anti-Mad1 antibody (1:3000) or anti-Mad2 antibody (1:1000), then washed and incubated with anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, 1:10,000). The blots were subsequently reprobed with anti-tubulin DM1A (Sigma-Aldrich, 1:3000, mouse) or anti-ATP synthase NN18 [22] (Sigma-Aldrich, 1:5000, mouse) as loading controls.…”