Difference between revisions of "Template:SUIT text D066"

Latest revision as of 12:55, 25 November 2020

The SUIT-029 O2 mt D066 is designed to evaluate the quality control of mitochondrial preparation. This protocol gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). SUIT-029 O2 mt D066 includes a control for contamination with other organelles containing ATPases (e.g., plasma membrane and endoplasmic reticulum.) In experiments with crude isolated mitochondria, tissue homogenates and permeabilized cells, it is expected to have active ATPases in the preparation. When ATPases are active, residual endogenous adenylates are phosphorylated to ATP in the LEAK state, which is recycled to ADP in isolated mitochondria contaminated by ATPases, which leads to an overestimation of LEAK respiration, when comparing L(n) and L(Omy). Therefore, it might be necessary to assess LEAK respiration in the presence of ATPase inhibitors, e.g. oligomycin. Additive effect of convergent electron flow through NADH- and NS-pathways is evaluated in the ET state.

The present SUIT-029 O2 mt D066 DLP file shows an application with imt, thom or pce in the pathway category NS. For using this protocol with other mitochondrial preparations or substrate/inhibitor combinations, a personalized DLPU can be created.

@@ Line 1: / Line 1: @@
-The SUIT-006 O2 mt D066 is designed to evaluate the quality control of [[Isolated mitochondria|isolated mitochondria]]. This protocol includes a control for contamination with other membranous organelles containing ATPases (''e.g.'', plasma membrane and endoplasmic reticulum.) In experiments with crude isolated mitochondria,tissue homogenates and permeabilized cells, it is expected to have active ATPases in the prepation.
+::::The SUIT-029 O2 mt D066 is designed to evaluate the quality control of [[mitochondrial preparation]]. This protocol gives information of the linear coupling control ([[LEAK respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET capacity| ''E'']]) with NADH linked-substrates ([[PM-pathway control state|PM]]). SUIT-029 O2 mt D066 includes a control for contamination with other organelles containing ATPases (''e.g.'', plasma membrane and endoplasmic reticulum.) In experiments with crude isolated mitochondria, tissue homogenates and permeabilized cells, it is expected to have active ATPases in the preparation. When ATPases are active, residual endogenous adenylates are phosphorylated to ATP in the LEAK state, which is recycled to ADP in isolated mitochondria contaminated by ATPases, which leads to an overestimation of LEAK respiration, when comparing [[LEAK state without adenylates|''L(n)'']] and [[LEAK respiration|''L(Omy)'']]. Therefore, it might be necessary to assess LEAK respiration in the presence of ATPase inhibitors, ''e.g.'' oligomycin. [[Additive effect of convergent electron flow]] through [[N-pathway control state|NADH-]] and [[NS-pathway control state| NS-pathways]] is evaluated  in the [[ET capacity| ET state]].
-When ATPases are active and residual endogenous adenylates are present in the sample, ATP can be consumed and converted to ADP, which will stimulate respiration. This may lead to an overestimation of LEAK respiration - [[LEAK state without adenylates|L(n)]]. Therefore, if the samples are contaminated with ATPases/adenylates, it might be necessary to assess LEAK respiration in the presence of ATPases inhibitors, ''e.g.'' oligomycin - [[LEAK|L(Omy)]]. This protocol can be used as a quality control of the [[mitochondrial preparation]].
+::::The present SUIT-029 O2 mt D066 DLP file shows an application with imt, thom or pce in the pathway category NS. For using this protocol with other mitochondrial preparations or substrate/inhibitor combinations, a personalized [[Export DL-Protocol User (*.DLPU)|DLPU]] can be created.
-In the DatLab software, SUIT-006 O2 mt D066 DLP file is provided for mt, tissue homogenate or for cells which were permeabilized before addition into the O2k-Chamber and for the category NS. For using this protocol with other sample preparation or substrate/inhibitor combinations, a personalized [[Export DL-Protocol User (*.DLPU)|DLPU]] can be created.