Talk:MiPNet12.15 RespiratoryStates: Difference between revisions

Revision as of 14:06, 3 July 2012

State4 vs oligomycin

Gyorgy Szabadkai (MD, PhD; London, UK) wrote:We have recently started to work on isolated mouse cardiac mitochondria on an Oroboros setup and would like to pick your brains on some puzzling results, see screenshot of traces:

State 4 respiration is very high, despite the very low uncoupled respiration when oligomycin is applied. What do we miss here?

Below is a short description of the protocols used: Cardiac mitochondria were isolated from a single mouse heart (0.1mg) using trypsin digestion (5mg/ml) and differential centrifugation. Fatty acid free solutions were used to avoid uncoupling mitochondrial respiration. All steps in the isolation were carried out at 4°C.Respiration was measured using an Oroboros respirometer at 37°C.

1) ADP titration: 0.25mg mitochondrial homogenate was added to a 2ml total volume (using miro 5 as respiration medium). Substrates used were pyruvate 10mM and malate 10mM. ADP 0.125mM added to achieve a State 3 respiration. After a steady State 4 respiration was achieved a further ADP 0.125mM was added. Max ADP (0.5mM) was finally added to check for saturation of ATPase at 0.125mM and subsequently oligomycin 2.5µM was added to assess Leak rate.

2) Effect of oligomycin on state 4 respiration: As before 0.25mg mitochondrial homogenate used. Oligomycin 2.5µM added after State 4 respiration achieved.

We would highly appreciate your comments on these findings.

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 == '''State4 vs oligomycin''' ==
-* ''Gyorgy Szabadkai (MD, PhD; London, UK) wrote'':We have recently started to work on isolated '''mouse cardiac mitochondria''' on an Oroboros setup and would like to pick your brains on some puzzling results, see screenshot of traces [[File:Szabadkai State4-vs-oligo.JPG|left|200px]] attached. Below is a short description of the protocols used. '''State 4 respiration is very high, despite the very low uncoupled respiration when oligomycin is applied'''. What do we miss here?Cardiac mitochondria were isolated from a single mouse heart (0.1mg) using trypsin digestion (5mg/ml) and differential centrifugation. Fatty acid free solutions were used to avoid uncoupling mitochondrial respiration. All steps in the isolation were carried out at 4°C.Respiration was measured using an Oroboros respirometer at 37°C.
+* ''Gyorgy Szabadkai (MD, PhD; London, UK) wrote'':We have recently started to work on isolated '''mouse cardiac mitochondria''' on an Oroboros setup and would like to pick your brains on some puzzling results, see screenshot of traces: [[File:Szabadkai State4-vs-oligo.JPG|left|200px]]
-) ADP titration0.25mg mitochondrial homogenate was added to a 2ml total volume (using miro 5 as respiration medium). Substrates used were pyruvate 10mM and malate 10mM. ADP 0.125mM added to achieve a State 3 respiration. After a steady State 4 respiration was achieved a further ADP 0.125mM was added. Max ADP (0.5mM) was finally added to check for saturation of ATPase at 0.125mM and subsequently oligomycin 2.5µM was added to assess Leak rate.
+'''State 4 respiration is very high, despite the very low uncoupled respiration when oligomycin is applied'''. What do we miss here?
+Below is a short description of the protocols used:
+Cardiac mitochondria were isolated from a single mouse heart (0.1mg) using trypsin digestion (5mg/ml) and differential centrifugation. Fatty acid free solutions were used to avoid uncoupling mitochondrial respiration. All steps in the isolation were carried out at 4°C.Respiration was measured using an Oroboros respirometer at 37°C.
+) ADP titration: 0.25mg mitochondrial homogenate was added to a 2ml total volume (using miro 5 as respiration medium). Substrates used were pyruvate 10mM and malate 10mM. ADP 0.125mM added to achieve a State 3 respiration. After a steady State 4 respiration was achieved a further ADP 0.125mM was added. Max ADP (0.5mM) was finally added to check for saturation of ATPase at 0.125mM and subsequently oligomycin 2.5µM was added to assess Leak rate.
+) Effect of oligomycin on state 4 respiration: As before 0.25mg mitochondrial homogenate used. Oligomycin 2.5µM added after State 4 respiration achieved.
-) Effect of oligomycin on state 4 respirationAs before 0.25mg mitochondrial homogenate used. Oligomycin 2.5µM added after State 4 respiration achieved.
 We would highly appreciate your comments on these findings.