mini-rotofor apparatus  (Bio-Rad)

Journal: Autophagy

Article Title: Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain

doi: 10.4161/auto.7.9.16436

Figure Lengend Snippet: Old and new BHMT forms. (A) Amino acid sequences (red) from BHMT forms associated with autophagosomal membranes, previously mapped by MALDI-TOF tryptic fingerprinting.39 Blue lines indicate amino acids thought to be involved in binding of the homocysteine substrate; yellow lines indicate an involvement in betaine binding and green lines indicate participation in catalysis.86 Grey lines indicate sites involved in dimerization (316–349) and tetramerization (381–407).87 (B) A frozen-thawed cytoplasmic extract (postnuclear supernatant) from rat hepatocytes was solubilized in an SDS-containing lysis buffer, fractionated by SDS-PAGE and immunoblotted with an N-terminal BHMT antibody. In addition to full-length BHMT (p45), several BHMT fragments (10–33 kDa) are detected. (C) Separation of BHMT forms by liquid-phase isoelectric focusing. A frozen-thawed cytoplasmic extract (postnuclear supernatant) from rat hepatocytes was fractionated by liquid-phase isoelectric focusing (LP-IEF) on a mini-Rotofor into 20 fractions of different pH values as indicated. Each Rotofor fraction was further fractionated by gel electrophoresis and immunoblotted with the N-terminal BHMT antibody. The figure is a composite of three separately stained blots from two different gels.

Article Snippet: The lysate (19 ml; ∼100 mg protein) was loaded into a mini-Rotofor apparatus (Bio-Rad) and electrofocused at 20°C for 5 h at 12 W. Twenty fractions (∼0.45 ml each) were collected, pH was measured in each fraction and a 50-µl aliquot was neutralized (with NaOH or HCl) and analyzed by immunoblotting with the N-terminal BHMT antibody.

Techniques: Binding Assay, Lysis, SDS Page, Nucleic Acid Electrophoresis, Staining