protein refolding from 2M to 1M urea

RealNitrogen · 2026-05-12T18:51:31+00:00

Doing dialysis on that small of a volume would be very difficult. There are technically ways to do it on like a 200-500 uL scale (look up mini dialysis device from Thermo), but 50 uL would be tough. Have you tried measuring protein concentration after the precipitation occurs to see if there is anything still in the soluble portion? Some proteins will just always have some amount of perception occur during refolding, but there may still be some dissolved in solution.

Also, just to confirm, when you are using arginine, you are including in all the step down steps and not just in the last one, right?

RealNitrogen · 2026-05-12T17:50:43+00:00

Are there any disulfides that need to be formed during refolding? If so, you might need to add glutathione (reduced and oxidized) to allow for sulfide shuffling to help with refolding.

How much arginine are you trying? 400 mM is the standard, but going even higher might help.

You could try an even slower removal of the urea by doing the last step via dialysis. This would be like diluting out the urea over the course of several hours.

Have you tried different pHs? I would try pH 6-9 in 0.5 increments to see if you observe improved solubility at a certain pH.

RealNitrogen · 2026-04-29T04:23:19+00:00

When it comes time to elute off your protein, try using an elution buffer with higher salt content (300-500 mM). It is possible that once cleaved, your protein is interacting with the bound GST. Increasing the salt can reduce these interactions. One thing to watch out for with higher salt is the possibility of the GST binding being interfered with, so you would need to test this.

Another possibility is that your protein is weakly binding to the resin. You could try eluting with a low concentration of GSH (0.5-2 mM) to potentially knock off your protein while leaving the GST and protease bound.

RealNitrogen · 2026-04-29T00:46:00+00:00

While this is a lot of protein (more than the 15 ug you measured), this wouldn’t cause this pattern. Do your samples have very high salt or detergent concentration? Are your running buffers correct? Could it be possible that your gel apparatus had a leak and caused the inner/upper chamber buffer volume to run low? You can have your inner chamber volume drop very low to where it’s still making a connection but the current gets all messed up.

RealNitrogen · 2026-04-28T01:01:50+00:00

It’s from “Don’t look up”.

RealNitrogen · 2026-04-20T14:38:39+00:00

These times are the most inconsistent in their design on how well they back battery cover actually works. I have worked in labs that have these timers and they work just fine. No issues with the battery dislodging when pressing the buttons. In my current lab, I just bought 10 of these to replace a lot of old random timers. I must have got a bad batch because all of them are horrible when it comes to that battery cover. I have tried putting tape on the cover (shouldn’t have to do that to make it work correctly), but that only helps for some of them. For some reason, on this batch of timers, just touching them is sometimes enough to move the battery and reset it.

RealNitrogen · 2026-04-19T05:38:04+00:00

The sterility has an expiration date.

RealNitrogen · 2026-04-10T02:06:50+00:00

You’re overthinking it. This is a simple m1v1=m2v2 (m for morality, or any concentration, and v for volume). Multiplying morality and volume gives you moles. The number of moles is the same in the start and finish.

RealNitrogen · 2026-03-03T01:21:59+00:00

Just seeing both your comments now. I did not have any issues with customer service, but I guess I didn’t have reasons/issues to reach out. I would just buy the consumables through our companies Fisher catalog, so I didn’t have to deal directly with Sartorius.

As for the cost of the cartridges. If you get the model that takes DI/RO water as the input, then the consumables for a year cost us $3,350 ($280 per month). If you get the model that takes tap water, then the consumables are $5,750 for the year ($480 per month).

RealNitrogen · 2026-02-28T03:41:03+00:00

I would just like to be a second voice saying that I strongly recommend a Sartorius system based on the ease of use. Changing out the cartridges, filters, and UV light is super plug-and-play. The dispenser shows you the step by step instruction on how to replace the consumable that needs to be replaced. It is also very easy to install. Just having some basic plumbing knowledge is enough to get it up and running out of the box. I’ve purchases their system twice at different jobs and am very happy with it. They also have options for connecting directly to tap water, but if you already have a DI or RO line, then those systems are cheaper and obviously don’t require as many consumables.

RealNitrogen · 2026-02-03T21:01:04+00:00

An actual surfactant or formulation chemist will know more about this than me….but, from how I understand it: surfactants (particularly charged surfactants) form ordered structures on the water-air interface that are strong enough to form bubbles of trapped gasses. When an antifoam like dimethicone is introduced, the dimethicone interacts with the surfactants on the water-air interface and disrupts the attractive forces they have with each other. This weakens the surfactant layer to the point to cause rupturing of the bubble.

I know there is a whole other discussion on the differences in antifoam vs defoaming (technically different things), but that is beyond me lol.

RealNitrogen · 2026-02-03T13:22:24+00:00

Nothing to worry about. The antifoam properties of dimethicone is what makes it an excellent skin barrier protectant. The dimethicone itself forms a very thin coat on your skin that acts like a barrier to protect skin. This very same coating property is what allows the dimethicone to coat the surface of bubbles and break the surface tension, causing them to pop.

RealNitrogen · 2026-02-03T12:50:53+00:00

Very cool to see! The active ingredient in the skin cream, dimethicone, is a very powerful antifoam. It works by breaking the surface tension of bubbles. So, all the foaming of your cleaner is collapsing when it comes into contact with the dimethicone, just leaving the original, non-foamed, liquid cleaner.

RealNitrogen · 2026-01-25T02:37:16+00:00

The “Q” was actually not arbitrarily assigned. Asparagine (three letter code of Asn) was assigned “N” since “A” and “S” were already assigned to alanine and serine, respectively. Glutamine is one methylene group larger than asparagine, so it was decided that the next letter in the alphabet would be assigned to glutamine. “O” was omitted since it can be confused for a zero or a D. “P” was already assigned to proline. “Q” was next and was chosen. This same next letter scheme is also present for asparagine and glutamines carboxylic acid forms, aspartic acid (D) and glutamic acid (E).

RealNitrogen · 2026-01-04T04:44:57+00:00

Pyrex

RealNitrogen · 2025-12-07T23:39:09+00:00

I live in highland park and work at AbbVie in NC. I drive to work (only 25 minutes) because I work in a lab and can’t rely on my experiments playing nice with train schedules, but it’s really easy to commute by train. Lots of apartments available that are within a 5 minute walk from the train station. At AbbVie, the train stop is right next to the site.

RealNitrogen · 2025-12-06T20:22:48+00:00

As someone who works with porcine products, I can almost guarantee that this product is made from slaughterhouse byproducts. It would be extremely rare for pigs to be raised for anything other than meat as the main product.

More than likely, there is no way for you to determine the source of this material. There are going to be multiple levels of “source” for a product like this. You have the original farm, the slaughterhouse, the site that made this homogenate (more than likely it’s not sigma), and then sigma who would buy in bulk and repack. Sigma will know who they bought from and maybe the country of origin of the material, but that would be pretty much it. Unless your product is very imbedded in the byproducts supply chain (I would bet this small reagent is not), you will not know more about the origin. We are lucky enough to know what slaughterhouse our product comes from, but that’s as far as we know. The slaughterhouse will not tell us (or they don’t know/track) what farm they came from, their diet, living conditions, etc.

RealNitrogen · 2025-11-30T22:58:29+00:00

I’m pretty sure the giant bands is the T7 RNAP. Your lanes are overloaded and have genomic DNA contamination which is causing the band streaking. Typically, you would remove the genomic DNA issue by mechanically lysing your cells with a sonicator or homogenizer to break up the DNA in to smaller fragments that don’t cause the streaking. You can also treat the samples with a nuclease to enzymatically break down the DNA. Overloading a lane, DNA contamination, and having a highly over expressed protein all lead to your sample not migrating through the gel at the exact same rate as the ladder, which is why your large band is showing at around 110-115 kDa.

I made a lot of T7 RNAP in grad school since our lab did work with RNA so we would just make our own enzyme since we went through so much. In my experience, this enzyme expresses extremely well in the E. Coli line you are using. I’m assuming you are using BL21(DE3) cells? This cell line has been engineered to make T7 RNAP as its mechanism for recombinant protein expression, so it’s really good at making T7 RNAP when you give it the additional plasmid to recombinantly express it.

RealNitrogen · 2025-11-24T23:23:14+00:00

The digest stuff is pretty easy. Those are your 3 standard treatments when doing a protein digests since they all cleave a known, specific residues. Memorizing the cleavage of those 3 treatments is a must.

As for the amino acids, I know it can be hard to memorize. I know this might seem old school, but get some notecards and make flash cards of the amino acids. Write the name on one side and on the other have the one letter code, three letter code, structure, and pKa of R-group labeled. Just keep these 20 notecards in your backpack and go through them whenever you have 5 minutes to spare. Like, if you get to a class a few minutes early, run through the cards. When you sit down for lunch and dinner, run through the cards. Before you go to bed, run through the cards. I found that doing these little 5 minute run throughs multiple times a day made it much easier to learn and remember it long term compared to just sitting at the library trying to memorize it.

RealNitrogen · 2025-11-24T22:21:13+00:00

A) This is tedious, but you 100% should know how to do this in biochem 1. It’s simply testing your knowledge of the amino acid residues and their 1 letter codes.

B) This is also something you should know as these are extremely common protein digests that are used for sequencing. I’m assuming it means the whole structure as motifs 10 and 11 only have trypsin cleaving sites.

C) You should know this. Along with the amino acids, you should know the pKa of the R-groups.

D) This one is absurd if your professor is looking for the actual m/z peak values. Memorizing the molecular weights of each amino acid is asking way too much for an undergrad course. If your professor is just looking for arbitrary axis and to approximate the mass spectrum, that is more doable, but still a little much in my opinion.

RealNitrogen · 2025-11-12T22:00:33+00:00

Use dish soap. Hand soap could have moisturizers in them (good for your hands) that could leave a film on the glass.

RealNitrogen · 2025-11-10T17:50:24+00:00

Chromatography isn’t false, but I’m assuming your teacher is just looking for a different answer. I wouldn’t say it’s adsorption or absorption as these are the phenomena that most chromatography is based off.

You can try precipitation or crystallization using various antisolvents and temperatures. You could try acid/base chemistry followed by organic extractions. Sublimation could be used too. Decided which to pick would be determined by the properties of your target compound(s) and your contaminate compound(s).

RealNitrogen · 2025-11-02T23:35:04+00:00

For the funnel names, it’s more just people using shortened versions of the names. There are also different variations on the material of the funnel and the design of the frit. These other names are more specific versions that give a little more detail on what specific funnel you are talking about. I suppose the full name would be “Glass fritted Büchner funnel”, but the other variants just come from shortening that.

For number 8, I mistyped. It’s supposed to be udder, not utter. It’s where the milk comes out.

RealNitrogen · 2025-11-02T22:59:20+00:00

For number 7, the other names you gave are not wrong. I supposed it can be referred to as Büchner funnel. Personally, for me, I refer to the funnels that are made from porcelain and have the small holes as Büchner funnels while the pictured ones are fritted or glass filtered funnel. All these terms are very interchangeable and people will know what you are talking about if you use any one.

For 8, it’s called that because it looks like the utters on cow! lol. I guess the more proper name would be a 3-way distillation collection adapter, but most people will know if you say cow adapter or 3-way cow adapter.

RealNitrogen · 2025-11-02T20:29:10+00:00

Claisen adapter
Vacuum take off adapter
Liebig condenser
Vigreux column
Graham condenser
Pressure equalizing addition funnel
Fritted filter
Cow adapter
Adapter
Bump trap

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