Electron Transport Complex IV - Science topic

I have been trying to find chemical assays to detect cytochrome c oxidase activity in a soil sample. While looking at the various methods, I came across these 5. However, I am not sure which of these methods would be the most effective while testing soil samples in situ. Since, all of these methods seem to use different chemicals, it would be a great help if someone could suggest which method to proceed with. Thank you.

Hi,

I'm currently working on examining the activities of mitochondrial respiratory chain complexes. I successfully measured complex I, II and III; but the assay doesn't seem to work for complex IV.

Its an assay on whole tissue lysate (mouse heart) and its measured by monitoring the absorbance at 550nm. I follow the protocol outlined in this paper.

The only advice I came across was to check that my cytochrome c is reduced. I tested that but it wasn't the issue.

If anyone has any ideas about what could be wrong with my assay, I would be grateful for any advice :)

Hello everyone,

I did a complex II+III coupled assay following the procedure described by AntoniBarrientos, in

"In vivo and in organello assessment of OXPHOS activities".

However I realized that I forgot to add complex IV inhibitor, KCN, to the reaction. Here is the protocol

1. The assay is performed at 550 nm following the increase in absorbance resulting from the reduction of cytochrome c

2. In 1 ml of medium containing 10 mM KH2PO4 (pH 7.8), 2 mM EDTA, and 1 mg/ml BSA add mitochondria (20–40μg of protein) or whole cells (3–10×104 cells), 240μM KCN, 4μM rotenone, and 0.2 mM ATP.

3. Add 10 mM succinate as the donor and incubate 10 min at 30 °C.

Start the reaction by addition of 40μM oxidized cytochrome c. Measure activity for 5 min. The addition of 10 mM malonate inhibits the oxidation of succinate.

The absorbance in my experiment decreased with time, instead of increase, that's when I realized I forgot to add KCN, and, the complex IV was still able to oxidize the cytochrome C, which was reduced by complex III.

So, my question is can this assay be considered as a complex II+IV coupled assay? Please provide any references for II+IV coupled enzymatic assay. I found only II+III and I+III.

Thanks,

Razia

Hello!

I want to do a aphid-barcoding and was thinking of using the whole aphid and the standard primers for insects: LCO1490 and HCO2198.

Now I wonder if I might get problems, if there's plant DNA inside the guts of the aphid.

With bigger insects I have just used legs or other parts without guts so far. Since plants use Cytochrome Oxidase too, I'm concerned a little bit.

Does someone have experience with it - and if I will have problems: will I have to dissect the tiny aphid or will I at least get different sizes of bands on a gel and could solve this with cutting the right one out?

Thank you very much in advance!

Best regards,

Christina

Near-infrared Spectroscopy (NIRS)

For a clinician, it is important to understand the basic principles of NIRS, to know the limits of use and to interpret them correctly. NIRS is a technique in which the amount of absorption by which chromium molecules [oxyhemoglobin (O2Hb) and deoxyhemoglobin (HHb), cytochrome-c oxidase (CCO), myoglobin)] is measured as NIR light passes through the tissues. Chromophore can also absorb NIR light in tissues. There are molecules called. These molecules have specific absorption rates which vary according to the oxygen concentration in the tissue. The amount of light absorbed by the tissues is directly dependent on the chromophore concentration. At least two different wavelengths for comparison of chromophore concentration in NIRS measurements should be used. These two wavelengths are commonly used in measurements as O2Hb and HHb used in the measurements show the greatest difference in absorption at light of 700 to 850 nm wavelength. While two wavelengths are used in the first produced devices, the accuracy rate of the measurements is increased by using multiple wavelengths in the devices used today. The basis of the NIRS working principle is the Beer-Lambert law, an optical physics law. According to this law, the light is absorbed according to the material it passes through. Spectral resolution spectroscopy, frequency have developed techniques for dependent spectroscopy and time-dependent spectroscopy. Determining the chromophore concentration level in benign and malignant nodules in thyroid nodules has become simpler with these methods. I am hopeful that a non-invasive diagnostic method will be developed by the departments that support this hypothesis without being done in the future.

last week i did cytochrome oxidase staining to find the barrels in rat barrel cortex and i could find clearly , now i am planing again and i want to add imidazole in the ctox soultion but i find many forms of imidazole and i dont know which one is good for cytochrome oxidase, please answer.

I just got a result from microgen that unknown insect species(probably some kind of gall midge) using mitochondria COI region after DNA sequencing, alignment and blasting.

It was recognized that unknown sequence is 85% similar to Rhopalomyia sp.

(BOLD:ACC5938 voucher BIOUG06875-F04 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial)

I don't know whether I can identify it as a Rhopalomyia sp.

Is there a well structured database of sequences of terminal oxidases? The different terminal oxidases have different oxygen affinities and I have been unable to find a specialized database of sequences associated with these enzymes. Some examples of terminal oxidases are aa3-type cytochrome c oxidase, cytochrome bd-quinol oxidase and Cytochrome cbb3 oxidases. I am particularly interested in terminal oxidases in bacteria and archaea. Thanks in advance.

Hello everybody,

I need to barcode some mites amplifying by PCR a stretch of their mitochondrial Cytochrome Oxidase I (COX I). But I don't have its exact sequence, as this is the question the obtained PCR product is expected to answer after sending this PCR fragment for Sanger conventional dideoxy DNA sequencing.

I've already used before a pair with degenerate primers with one of them containing a stretch of 6 consecutive inosines and it (fearfully!!!) worked out just fine, as published here:

The calculated degeneracy of such primer is an elevated number of 4096 (4x4x4x4x4x4) and it worked out fine in spite of it.

But now I need to amplify this COI fragment without knowledge of its exact sequence. I've already aligned the closest available species with publicly available sequences in order to find the most probable degenerate primer able to amplify any of them (at least in theory).

I've been aligning these sequences searching for a common stretch among them with the lowest possible degeneracy that's well below 4096, actually equal or below 300.

At last here comes my question, which number would be roughly the top possible for a degenerate primer to amplify a single DNA product? 500? 1000?

Is it much different for these bases to be scattered along the DNA primer instead of a single long consecutive stretch of degenerate bases (such as the 6 consecutive inosines I've already used successfully)?

Thank you all in advances for the attention, and also for the patience needed to read such long considerations.

There were same DNA templates from spiders. However, I found it is easier to do PCR and sequence successfully in mitochonarial genes like COI (Mitochondrial Cytochrome Oxidase I ), 16S (16S ribosomal RNA) and 12S (ribosomal RNA) than nuclear genes like H3 (histone subunit 3 protein coding gene), 28S and ITS2 (internal transcribed spacer 2).

In this phenomenon, can I exclude the problem of DNA templates and focus on the nuclear genes part? 

BTW, i asked a colleague who research on fishes also has met this problem. 

I attached the problenatic nuclear gene 28S sequencing data as the following files, it always occurred repeat A or T and then failed to get a right result. Please help me to solve this tough problem, many thanks.