Igor V Gorbenko

Siberian Institute of Plant Physiology and Biochemistry | SIPPB SB RAS · Department of Cell Biology and Bioengineering

Greetings, dear colleagues! Do you know any technique utilizing some ~ordinary molbiol lab reagents (not the commertial kits for isolated mitochondria staining) that will allow isolated mitochondria staining? Maybe SYPRO Ruby will work, since mitochondria do possess a high protein amount? DAPI to stain mitochondrial DNA (though in it didn't work to stain mtDNA, but maybe due to high plastids and nuclear fluorescence?)?

Or EtBr (though in authors emphasize that only stress-induced granules can be stained with EtBr)?

Or some dyes that are capable of staining lipids? Rhodamine 6G?

Or maybe the methods of in-gel activity staining would work, e.g. mixing mitochondria with NBT/BCIP substrate and supply with NADH? Will formazan precipitate inside of mitochondria?

Or the TTC test - since mitochondria is enriched with dehydrogenases?

Any ideas?

Greetings, dear colleagues!

Our team conducts research on newly discovered SIRC elements in plant genomes ( , which are thought to be MITE transposons losing inverted repeats products, which could influence genome regulation) using bioinformatics, and we plan to conduct experimental molecular biology studies to elucidate the functions of SIRC. The problem is - our team is specialized in molecular bology experiments aiming to reveal the functions of genes, not non-coding DNA elements. That's why I want to ask your expert opinion - what experimental techniques would help to reveal the functions of abundant DNA elements of repetitive nature?

What comes to mind is the creation of mutant lines without several of these elements, but such experiments are too large-scale and can last for years, which is too complicated at the moment.

Another technique that comes to mind is the amplification of certain sequences and examination using circular dichroism spectroscopy to reveal whether given elements have unusual secondary structure like G-quadruplex of triplex DNA etc that could influence processes of genome transcription or replication.

And one more - we thought it could be possible to capture and identify plant proteins that specifically recognize SIRC via some modification of EMSA (electroforetic mobility shift assay) method. Unfortunatelly, up to date we didn't find any mentions of EMSA variant that uses not single purified protein, but whole DNA-free nuclear lysate, with subsequent identification of binding proteins via MALDI-TOF.

What other in vitro experiments could be useful?

Greetings. Probably the question is not complex at all, but can't find an answer.

If I have RT-qPCR data of gene expression in a sample with multiple analitycal replicates - to compare it to data obtained in other experiments I need to normalize the expression of genes of interest to the expression of reference gene (which is constitutievely expressed)..

How to perform it if there are replicates and expression of both genes of interest and reference gene are in a form of Expression and Standard Error of the Mean?

Is there a formula to adjust GOI SEM using RG SEM?

Dear colleagues! I have an issue with purified arthropod mitochondria. I am used to work with plant mitochondria, tried a bit with mammalian, and I roughly understand how the pellet of 100 mcg of mitochondria (by protein content) should look like.

The problem is - arthropod mitochondria pellet that is measured to be of 100 mcg by Bradford looks 5 to 10 times bigger. Also when I solubilize that mitochondria using proper detergent amount - I see that the pellet lefts are really big.

My assumption is that these arthropod mitochondria membranes are, like Drosophila's, composed mostly of mono-unsaturated fatty acid residues and thus are much more detergent-resistant than typical mitochondrial membrane.

Are there any methods to overcome this?

Dear colleagues!

We are trying to isolate mitochondria from conifer plants. The regular Percoll step gradient isn't working due to the percularities of the material, so we want to try mixed Percoll-Sucrose gradient.

What should be the sucrose concentration of the steps? The percoll works better with steps 50%, 25% and 18%. Wanted to try this:

1. 18% Percoll 0.25 M Sucrose

2. 25% Percoll 0.5 M Sucrose

3. 50% Percoll 1.2 M Sucrose

But this cannot be done due to excess sucrose that won't fit in volumes (for 10 ml step we need to add 5 ml Percoll, 2 ml of Medium x5 (which is already pretty dense) and somehow add 3.2 g of sucrose - seemes unreal).

I wonder if we need to try to preserve the osmolarity of the steps and equalize it with another sugar (Mannitol?)? What do you think?

We isolated mitochondria from freshwater amphipods and found that being disperged throughout the media in a tube - mitochondria soon starts to form flakes and aggregates.

We thimk this is also affecting Bradford assay for mitochondrial protein - the values are 3-4 times lower than could be estimated by eye. Digitonon and DDM also poorly solubilize mitochondrial membranes.

Dear colleagues!

We are currently conducting protocol adaptation for isolation of mitochondria from freshwater amphipods. The isolation medium was from the protocol of mitochondria isolation from shrimp (Sucrose 0.125M, D-Sorbitol 0.375M, KCl 0.15M, EGTA 0.001M, MOPS 0.02 M, and 0.5% of BSA), and the wash medium with the same content except for lower concentration of EGTA - 0.025 mM.

During the isolation there was very strange upper phase after the second centrifugation - bright red precipitate on the upper side of a tube. Probably some pigments.

The mitochondria precipitate was heterogenic - the most heavy part of beige color (obviously mitochondria) and the other part of orange - that time thought to be mitochondria accidently dyed with pigment.

Then we conducted Percoll gradient centrifugation (the self-forming gradient, 30% Percoll) and expected mitochondria to be in relatively low part of a gradient. On the contrary, after the centrifugation (40000 g, 60 min) the gradient contained mitochondria layer only 5 mm from the sufrace (it was 38 ml tube). Instead being cloudy, the layer seemes like some sort of aggregates.

After the washing procedure entire mitochondria fraction was bright orange.

Next we tried to measure the protein content with Bradford method and found that mitochondria, being finely dispersed throughout the media, soon starts to aggregate and form flakes.

Bradford works poor - the aliquotes that shows 100 mcg of mitochondrial protein after centrifugation were a pellets like 300-400 mcg. So I propose that the detergent in Bradford is somehow insufficient for total solubilization of mitochondria.

Furthermore, when we conducted detergent solubilization procedures (digitonin and DDM) - after the procedures the pellets of unsolubilized mitochondria were almost like before solubilization. Plus, the DDM had 3 phases instead of 2: the lower unsolubilized mitochondria pellet, solubilizate interphase and flake-like bright orange upper phase seemed to be the pigment.

Our thoughts are: in amphipods body there are some agents that results in mitochondria aggregation and subsequent partial detergent resistance. Are there some procedures that we can add to remove them?

For the isolation we used full body of amphipods. Maybe the aggregating agents were in their gastrointestinal tract.

Also, we want to try discontinious Percoll but unsure about the steps - the only paper that I found on animal mitochondria discontinious Percoll gradient centrifugation was rat brain mitochondria (Sims et al., 2008), but not sure if it is applicable for crustaceans.

Greetings!

I have an issue that drives me crazy this evening...

I have a list of gene vectors, downregulated in different transgenic plants and I want to make a Venn diagram to visualize it and to show the intersections between plants.

But! The results from any package I used (in R) gaves me something like this (the uploaded picture 1)...

What's bothering me:

1. The numbers on "clear" (not intersected) parts of a diagram are lower, than the gensets I have. And I tried to use factor instead of character vectors, to remove possible duplications, to remove symbols (like space) that could cause software misunderstanding - all gaves me nothing... same result.

2. The intersection of vectors is not true - on the picture you can see that the intersection of 2 datasets (of 365 and 154 genes) - is 1133 genes!! How could that be?

The manual usage of intersect function on the same dataset gaves pretty correct results.

Maybe I am misunderstanding about Venn diagrams? Because in a web I found many examples of such strange mistakes - on the second picture from Datanovia you can see that the intersection of the red elliplse (of 58) and yellow (of 144) is 66!

It seemes logical to me that the intersection of 2 vectors cannot be greater than the length of a smaller vector. What am I doing wrong or misunderstanding?

Dear colleagues! I've tried to estimate the histone variants enrichment of several arabidopsis genomic sites but every time my enrichment result looks like the added screenshot. What am I doing wrong?

The running function is:

enr.df <- enrichment(query =gr_list[[1]], catalog = anno, shuffles = 24, nCores = 12)

anno is remap2020_histone_nr_macs2_TAIR10_v1_0.bed,

catalog is a GRanges of sites of interest of A.thaliana (all 5 chromosomes).

There are no warnings after function execution.

We try to use RepeatMasker for de novo annotation of Arabidopsis transposable elements but it seemes that DFAM database don't have plant-specific TE hmm profiles... and we don't have the access for giri repbase. Is there any other public-available databases?

Good day! I have a list (~10000) of unique DNA sequences about 10-20 bp.

I want to find out if they could evolve from one or several sequences, or emerged independently.

Some of the sequences have similar motifs and could be aligned, others haven't at all - so I can't just perform MSA and make a tree - the distance matrix contains many NAs.

I've tried using principal components analysis on k-mers (1-4) frequencies but it gives me nothing - the frequencies form one dense cloud of points with PC1 that have only ~4% explained variance.

And I found that universalmotif R package is capable of performing similar analysis using motif_comparison(), so I converted the sequences into sequence motif format (one for each), but when tried it on a short set of data - found that the algorithm works in a very strange way on list of motifs created each from only one sequence. Different methods gives the same result (added tree to the question) - the sequences that are different are placed near instead of sequences that are someway similar...

Good day!

I'm using the BioRad PROTEAN II XI chamber and gradiend polyakrylamide gel - 3.3%-20%. I used to run the gel overnight at 105-110 V, but this time got the "Error no load" after ~6 hours of run. All the connections are fine so - the problem i think is low conductivity of the gel/upper buffer/ lower buffer - but all was made using recipes that I successfully used before...

Dear colleagues! We plan to isolate mitochondria from freshwater amphipods, but didn't find any methods in literature - the closest found was the method of isolation from whiteleg shrimp Litopenaeus vannamei.

The problem is - the amphipods are quite small - around 1 cm long, so it's hard to isolate the gut before mitochondria isolation.

Will it work if we use just the sample of 10 g (or is that too much?) of amphipods and blender to homogenize it in isolation medium? Or it is crucial to select only some parts - for example only the amphipods legs and antennas?

P.S.: we do not have chitinase, nor the chance to get it in time.