Question

Draw western blots to show lysoso mal contamination in a mitochondrial fraction after density gradient centrifugation. 1. Decide how many western blots and indicate the lanes and the antibody that you will be using I to probe these blots. Draw bands clearly to indicate intensity. Circle the band that indicates contamination. 2. 3.

Answer 1

Here, two markers should be used for mitochondrial fraction and lysosomal fraction. For mitochondrial fraction, you can use succinate dehydrogenase and for lysosomal fraction, you can use b-galactosidase. When you proceed western blot antibodies you will be using to probe these blots will be anti succinate dehydrogenase (size: 70 kDa) and anti b-galactosidase (size: 116 kDa).

In lane 1, you should provide protein marker, in lane 2, you should provide positive control for mitochondrial fraction, in lane 3, you should provide positive control for lysosomal fraction and in lane 4, you should provide your test sample, where you isolated mitochondrial fraction. But in this case, you have lysosomal contamination. So, here you will expect two bands, one for mitochondrial marker and one for lysosomal marker.

- - - - - - - - Ő Marker - (Ера) Mit. Irac Lys frac Test sample 200 140 b-galac a tosidan 1 Succinate * b-galactosidase contamination + Succinate dehydro derydro genane genase /