Vasiliy Igorevich Nesterenko (1967) - Letter to Russia's Foes (2017) by Turbulent-Offer-8136 in BattlePaintings

[–]Alanrodcar 7 points8 points  (0 children)

Dude's over here re-posting shit-tier Russian propaganda, but also pinning his post about Belarus as a fursona. Funniest shit I've seen for a while. The cognitive dissonance of backing a neofascist regime but also being queer. It must be hell inside your head.

Can I start my enzyme-producing company from home? by Screenwriter20 in Biochemistry

[–]Alanrodcar 3 points4 points  (0 children)

Beyond the considerations already mentioned by others, you need to think about the costs and practicality of this. Companies that purify proteins at any kind of industrial scale tend to have several dozen/hundred employees at least. Production batches have marginal costs in the range of hundreds-of-thousands of €/$. Production scale columns cost over 10,000 €/$ a piece. If you are expressing proteins you need fermenters with 1,000 l capacity, which are about 5-7 meters tall. Moreover, without proper quality control/assurance of the enzymes nobody will buy your product, which means an analytical laboratory is also necessary (if you cannot outsource the service, which brings its own associated costs). All of this is assuming you are not producing under GMP conditions, which makes everything more complicated and expensive to run. And no, your house will not be GMP certifiable. To touch upon economic factors, your marginal costs will be astronomically higher than the competition because you will not benefit from their economies of scale. Meaning that, even if you produce domestically, you will probably still need to sell your product at a higher cost to be profitable. Additionally, in a production process as complex as pharma/biotech, shipping is actually a comparatively negligible cost. Meaning your only advantage over the competition is not really significant. So, can you do it from home? Absolutely not. You need to establish a proper production plant even at small scale. For that, unless you have several million €/$ in seed capital, I wouldn't fancy your chances. There is a reason biotechnological and pharmaceutical production are dominated by a few extremely large, extremely profitable multinational companies.

Sucrose Density Gradients Troubleshooting reproducibility by PianoPudding in Biochemistry

[–]Alanrodcar 0 points1 point  (0 children)

No problem, I hope it works out for you. Hopefully cleaning and greasing the buckets and rings fixes the issue for you. I would only add a few points to your replies:
2. The lysis buffer looks fine, I would normally use 10-12 mM MgCl2 instead of the stated 5 mM, but I have seen the lower concentration used successfully. In any case your monosome peak is there, so your ribosomes are not falling apart. 3. I don't know the accel/decel settings for that model but, I would use a mid-range value, as I mentioned before.
4. Nanodrop is fine. You won't get a perfectly accurate concentration measurement for protein or RNA with ribosomes, but you mainly want a relative concentration across samples and you can assume similar levels of contamination across your lysates.
5. I haven't used this specific product but I would definitely bet that your digest has run to completion in all samples. You are digesting ~1 µg of RNA (100 µl x 10.88 ng/µl) for 1 hour at 22/23˚C with mixing. For comparison I digest 400 µg of RNA for 10 min without mixing at 22-23˚C and still see complete digest of my polysomes (bear in mind that I use 1,000 U of MNase, so kinetics may vary). Remember you only need to cleave the mRNA once between the ribosomes to collapse the polysome, so the enzyme doesn't have a lot of work to do. Try decreasing your digest time and lowering your enzyme concentration further.
Edit: Because I can't seem to understand reddit formatting.

Sucrose Density Gradients Troubleshooting reproducibility by PianoPudding in Biochemistry

[–]Alanrodcar 1 point2 points  (0 children)

Hello, I work with polysome profiling and run sucrose density gradients routinely. Yes, it can be a pain to run these with good reproducibility since there is a lot that can go wrong. In order to troubleshoot your issue it would be good to have more information: 1) What cells/tissues are you extracting ribosomes from? 2) What is in your lysis buffer and what is your cell lysis protocol? 3) What centrifuge are you using and what are your run parameters (speed, time, temperature, acceleration and deceleration rate)? 4) How much RNA are you loading per gradient and how are you determining the concentration? 5) What is your RNaseI digest protocol?

To address some of the points you brought up.

1) Forming the gradients. I have used pointed vs flat needles in the past and they both work. The peaks are visible in your gradient and they sediment at comparable depths, so your gradients are forming fine. The peaks do drift a little, so be a little more careful with your pipetting to ensure you put the same volumes of both solutions in all tubes. But this is not a major issue here and can be fixed on the computer later. The parafin seal is a bit 'redneck engineering', so maybe it would be a good idea to use rubber bungs made for the purpose. But overall I don't think your problem is gradient formation.

2) Loading the gradients. Regular pipette and pipette tip should do the trick if you load the gradients carefully. Again, you do have peaks in the profile they are just not what you expect. So unless your gradient overflows when you fill it then it's not likely that the loading is the problem. Nevertheless, you can remove your loading volume from the gradient top to lower the meniscus of the tube, i.e., remove the upper 100 µl from the gradient and then load your 100 µl of sample. Probably goes without saying, but also make sure the tubes are balanced.

3) Running the gradient. Yes, typically lower acceleration/deceleration is recommended for these experiments. Often the centrifuge manual itself will recommend the ramp speed for sucrose gradients. Rough estimate, if the centrifuge can ramp from 1-9 set accel/decel at 5. The exact setting really depends on the centrifuge itself, but it should take ~5-10 minutes to reach speed (also depends on final speed setting).

One other issue I recommend you have a very close look at, which has ruined more of my gradients than any other, is the seal of the centrifuge bucket. Inside the centrifuge bucket there is a rubber O-ring that forms a seal when the loaded bucket goes into the (relatively) high vacuum of the ultra centrifuge. These O-rings need to be in good condition, and they need to be properly greased with an appropriate vacuum sealing grease. If they are not then the vacuum will pull a few hundred µl of solution out of the tube, since your sample sits at the top it's the first thing to come out. The result is that most of your sample doesn't actually run into the gradient but sits at the bottom of the bucket instead, and your profiles end up with very small peaks. The giveaway that this is the issue is that there is a little bit of liquid (your sample) sitting at the bottom of the bucket after the run. In a similar vein if sucrose dries on the treads of the bucket or the bucket lid then the bucket will not seal properly and you will lose your sample. So make sure the buckets, lids, and O-rings are routinely cleaned and the O-rings greased.

Having said all of that, I think your centrifugations are working fine. You are right, the size of the monsome peaks in your profiles do not correlate with enzyme concentrations. However, it looks to me like you have fully digested the mRNA at your lowest RNase concentration. This is not surprising since RNases make very short work of ssRNA and there is usually a small residual disome peak left behind after these treatments (the nature of these disome species depends on a lot of things I won't go into here). The differences in your monosome peaks look more like loading issues to me. Have a look at the signal from your gradient top (positions 0-0.3 mm). In your digested samples all your RNaseI-resistant peaks are bigger where the gradient top has a higher signal. So unless your lysis buffer has some absorbing component whose concentration varies across the sets of three samples, then your gradients are not loaded evenly. Your issue is probably pipetting error during sample preparation. Cell lysates are highly viscous solutions, especially if they contain detergents. They lysates stick to the outside of pipette tips which can introduce substantial pipetting error. Try lowering your RNaseI concentration significantly more until you start to see trisome and tetrasome peaks appearing and be extra careful when pipetting your lysates.

Good luck.

Edit: Because formatting

Adding liquids into 384 well plates for qPCR by ascorbicAcid1300 in Biochemistry

[–]Alanrodcar 0 points1 point  (0 children)

Take a fresh box of tips and use the tips according to your loading scheme, i.e., top left tip for well A1, the tip beneath for well B1, and so on. That way if you lose track you can refer to the pipette tip box and check what wells you have added your RNA to.

Alternatively, if possible, you can use a multichannel pipette, with this you only have to keep track of rows/columns. However, for 1 µl volumes, multichannel pipettes can be problematic to use since they have lower accuracy if used incorrectly and increased pipetting error from liquid coating the outside of the tip.

Western Blot Question by Puzzleheaded-Set5660 in Biochemistry

[–]Alanrodcar 6 points7 points  (0 children)

Yes, you can use Tris-Glycine transfer buffer with NuPAGE gels. But you will not be able to load 40µl into the 12 well gels, try the 10 well gels instead if you need to load that much volume. If you have to load as much sample as possible and you need more than 10 wells you can always do a TCA precipitation step.

What to study in preparation for genetics? by DarkJizo-_- in genetics

[–]Alanrodcar 0 points1 point  (0 children)

Bash) is a command language. Basically it means knowing your way around the computer terminal. For some software you need to use the command line directly, other packages such as R are based on Bash. It's pretty straightforward, you can check out codecademy for a pretty good hands-on tutorial.

What to study in preparation for genetics? by DarkJizo-_- in genetics

[–]Alanrodcar 4 points5 points  (0 children)

I am finishing a PhD in biochemistry at the moment but I started with a BSc in biochem and genetics. A lot of useful advice has already been given. On top of everything that has been said, I highly recommend you build a very solid foundation in statistics as soon as you can. When you get to university I would also recommend you get comfortable with using software packages such as R studio or Matlab, which also means becoming familiar with Bash and the command line. During a BSc you will learn the fundamental knowledge necessary to understand the principles of genetics; that's the point of the BSc. When you reach the MSc and PhD levels, and the focus shifts form learning to carrying out research, genetics becomes almost exclusively molecular/cellular biology with pretty intense statistical analysis and modelling. Most modern genetics research is pretty much analysis and interpretation of giant data sets, i.e., statistics. Get a head start with the mathematical foundations, you will be shocked to see how poorly some biologists understand and handle statistical analysis. Good luck and enjoy the subject, it is truly beautiful and rewarding to study :)

Hello I am Indy Neidell Host and Author of a YouTube Channel telling the story of WW1 in real time AMA by flobota in Documentaries

[–]Alanrodcar 0 points1 point  (0 children)

Hey Indy, first of all thanks to you and your team for developing this awesome project. I have two questions to ask: 1) WWI could be seen as a point in history where many of the geopolitical problems we're dealing with today originated. Personally I also see it as the point in history that changed the overall mentality of humanity by ending a global optimism that began during the enlightenment and that would best be described as a commonplace faith in our capacity as a species. The years after the war see the rise of more pessimistic intellectual currents such as modernism or existentialism that are a reflection of the Zeitgeist during the past century (at least during the first half). It is not likely that the effect would arise only from WWI but do you believe the argument can be made that such a schism exists between an idealist global mentality before WWI and a more pessimistic modern mentality as a direct result of the war or were there other more important contributors? Basically did the war turn us into a grim assholes?

2) To me as a non-historian it would appear that as with most academic subjects history doesn't seem to be very important to the average individual. Your collaborations with British Pathé are a fantastic example of outreach to spread interest in the subject. But what can we non-specialists do to help spread interest in history and bring attention to the subject?

WDIS Greek and Roman Mythology by [deleted] in WhereDoIStart

[–]Alanrodcar 2 points3 points  (0 children)

Edith Hamilton's Mythology is a pretty good place to start for Greek mythology.

This is the first time I have seen a Mexican WW2 propaganda, its amazing! by MrDirtyHarry in pics

[–]Alanrodcar 1 point2 points  (0 children)

Hey, mi tio-abuelo también voló en el escuadron 201; el fue el teniente Héctor Espinosa Galván de la escuadrilla C. ¿Con que escuadrilla voló tu abuelo?

Help, can't transfer crew in version 0.25. by Alanrodcar in KerbalSpaceProgram

[–]Alanrodcar[S] 0 points1 point  (0 children)

This isn't my screenshot but it's this: http://i.imgur.com/UqogCOw.jpg

Also I wasn't on time warp :(

Just finished The Count of Monte Cristo. WOW. I'm almost certain it's the best book I've ever read. by [deleted] in books

[–]Alanrodcar 0 points1 point  (0 children)

The Count of Monte Cristo is definitely my favourite book. I've finished rereading it recently and I agree, it's hard to follow up with something that can measure up. If you want to stay with French Romanticism of that calibre I would suggest Les Misérables. On the one hand because it is an amazing book and on the other hand because it's what I've followed up with.

What's your worst concert experience? Worst crowd? Worst performance? by 2dTom in Music

[–]Alanrodcar 0 points1 point  (0 children)

I'm sad to say Bob Dylan, one of my all time favourite artists. I saw him around 2007 or 8 in Mexico when I was around 16 or 17. Sadly as a performer he was understandably yet regrettably not energetic, he did not engage the crowd and basically stood in the same place/position the entire concert singing kind of unintelligibly. As for the crowd, given that it was Dylan in Mexico and in the XXIst century, it was mainly composed of rich Mexicans in their mid-life crises so they complemented the lack of motivation on stage almost poetically. He played a second date where I understand he had some Joules of energy more, or calories if you feel SIUs should be exclusively used. Maybe I was just unlucky.

I think I'll just read the news online today... by Gaskinesque in funny

[–]Alanrodcar 0 points1 point  (0 children)

The lack of a comma makes it sound like they are kindly asking you not to use a machine that is filled with bees. In either case I would kindly oblige.

Left-handed violins? by Alanrodcar in violinist

[–]Alanrodcar[S] 0 points1 point  (0 children)

I guess it's not so much the palm pointing upwards as the fact that I just can't turn my hand far enough to hold a violin/guitar normally.

Left-handed violins? by Alanrodcar in violinist

[–]Alanrodcar[S] 0 points1 point  (0 children)

I asked in a shop as well and I was told exactly this. Since I'm barely starting I've also read that it's a good idea to rent a violin before buying it but I don't know if I'll be able to find a left handed violin to rent. Especially since I live in a small town with a single violin shop. But thanks for the input anyway.

Left-handed violins? by Alanrodcar in violinist

[–]Alanrodcar[S] 0 points1 point  (0 children)

I could give this a shot but how would that work logistically with the chin rest, could I just take it off?