Proteinase Puns

I have been battling autoimmune symptoms for over 4 years without a diagnosis. First doctor said possibly Wegener’s GPA. My third rheumatologist said possibly MCTD and she’s wanting me to go to Mayo. I am saving up for Mayo but have been told they have to also accept your case so even with $, may not be a sure thing. My symptoms match both Wegener’s and MCTD. I have had no less than 3 blood test a year for the last 4 years. Each time I have tested positive for PR3 antibodies. The highest level was 4.5H and lowest was 2.3H. I have only tested positive for ANA twice, with an ANA pattern of Nuclear, dense fine speckled. My inflammation markers were really high when I was first tested but normal every other time since. My WBC, creatinine urine, protein total and a few other markers are low most of the time but right under the normal range, never extremely low so they don’t get looked into. I am tired of feeling like crap everyday and the fatigue and brain fog is causing me daily grief with my job and life. I can’t be treated without a diagnosis and just visited my 4th rheumatologist who said it was all in my head even though arthritis in my fingers are starting to twist them. I don’t know where to go for help or what my next step should be. I’m getting depressed and feeling really hopeless with no help or direction.

Hi everyone,

Anyone currently using Proteinase K recombinant in Bulk? How much are you paying? Curious to know.

Thanks,

A protein that digests proteins, but does not get digested by itself.

I'm troubleshooting my RNA extraction these days because the particular organ that I need to isolate RNA from usually has low purity (around 260/280 of 1.6-1.7 by nanodrop). It does works in the downstream qPCR, but some samples fail to give strong housekeeping gene expression even when their RNA concentration is sufficient, so I'm wondering if the poor quality is the issue.

As for this tissue, its a very small organ that doesn't give a lot of RNA, and its generally impossible to dissect it out without also getting a lot of ECM into the tube as well (usually its like a gel of ECM surrounding the smaller tissue), which I think could be a source of the low quality because there is likely to be a lot of ECM proteins. For comparison's sake, bigger organs that have much more cells:ECM tend to give me 260/280 between 1.8 and 2, so I think it probably is the ECM proteins rather than genomic DNA or some other problem with the Trizol protocol itself.

My lab only has proteinase k as a part of DNA extraction protocol in which the sample is heated overnight, which would probably be too much for the RNA to avoid degredation. Does anyone use proteinase k with trizol, and if so, at what step do you use it? Before the phase separation or after the extraction is over?

proteinase K digestion of the protein

Proteinase K is a member of the family subtilisin, typically a proteinase. The molecular mass of the proteinase K is ~28, 930kDa. The enzyme is typically a protein which is made up of the long chain of approximately 278 amino acids. The proteinase K digests mainly the hydrophobic amino acids ( both aromatic as well as aliphatic). The cofactor, calcium ion provides stability to the enzyme, However, it cannot increase the activity of the reaction. Read more on proteinase K DNA extraction, the complete protocol and lot more here: Proteinase K DNA extraction method

Hello, I am doing an LPS separation gel, and in the protocol it calls for 1 microlitre of proteinase K into my samples, and sit for 1 hr at room temp before loading onto the gel. My PI asked me to look up what units are, but on the NEB website, it gives me a description about digestion of urea denatured hemoglobin. I am trying to figure out what units/ml represents concentration wise. Thanks.

I just wondered if buying Proteinase K that claims to be RNase free was really necessary - isn't it all naturally going to be RNase free? Are we just victims of some sort of advertising strategy - like gluten free Angus beef (ignore arguments over processing), or is there really something to this like potential RNase concentration levels?

Fact or myth, can you explain further and provide some good detail?

Thanks!

So I'm trying to partially proteolyze a protein with Proteinase K and then using SDS PAGE. It's behaving really strangely though. I've tried different weight ratios of protein:ProK, and maybe I'm using too much proteinase K, but if I mix my protein with ProK and then IMMEDIATELY add loading dye with SDS/bromophenol blue/mercaptoethanol I still see just as much cleavage as I do when I let it sit for 10 min or 30 min at 37C. I find that really strange, since the company I bought it from said it can take 1-24 hours at a concentration of 50-100 ug/mL and I'm using less than that (4 ug/mL and a weight ratio of 1:50 (PK:protein)). I'm working at pH 6 using an acetate buffer (my proteins are more stable at pH 4 actually but that pH is too low for PK. Saw almost no change at a weight ratio of about 1:2 protein to PK) What gives?

I'm currently using a Qiagen DNA Minikit to extract DNA from human dermis for quantification using a picogreen assay. Our results going by their IFU were pretty terrible, so we have modified their protocol. However, this has caused us to use an obscene amount of their proteinase K preparation in the tissue digestion step, which is expensive.

As such, we are trying to buy lyophilized proteinase K to save money. However, our homebrewed proteinase K preparation fails to digest minced tissue in a reasonable amount of time. Does anyone know of a buffer that will maximize proteinase K activity for the purposes of tissue digestion? I've googled and seen different preps using tris, triton, EDTA, and CaCl2, but does anyone have a homecooked recipe they prefer? Would be much appreciated, thanks!

I don't want to step on anybody's toes here, but the amount of non-dad jokes here in this subreddit really annoys me. First of all, dad jokes CAN be NSFW, it clearly says so in the sub rules. Secondly, it doesn't automatically make it a dad joke if it's from a conversation between you and your child. Most importantly, the jokes that your CHILDREN tell YOU are not dad jokes. The point of a dad joke is that it's so cheesy only a dad who's trying to be funny would make such a joke. That's it. They are stupid plays on words, lame puns and so on. There has to be a clever pun or wordplay for it to be considered a dad joke.

Again, to all the fellow dads, I apologise if I'm sounding too harsh. But I just needed to get it off my chest.

wouldn't the SDS/boiling get rid of any proteases?

Alot of great jokes get posted here! However just because you have a joke, doesn't mean it's a dad joke.

THIS IS NOT ABOUT NSFW, THIS IS ABOUT LONG JOKES, BLONDE JOKES, SEXUAL JOKES, KNOCK KNOCK JOKES, POLITICAL JOKES, ETC BEING POSTED IN A DAD JOKE SUB

Try telling these sexual jokes that get posted here, to your kid and see how your spouse likes it.. if that goes well, Try telling one of your friends kid about your sex life being like Coca cola, first it was normal, than light and now zero , and see if the parents are OK with you telling their kid the "dad joke"

I'm not even referencing the NSFW, I'm saying Dad jokes are corny, and sometimes painful, not sexual

So check out r/jokes for all types of jokes

r/unclejokes for dirty jokes

r/3amjokes for real weird and alot of OC

r/cleandadjokes If your really sick of seeing not dad jokes in r/dadjokes

Punchline !

Edit: this is not a post about NSFW , This is about jokes, knock knock jokes, blonde jokes, political jokes etc being posted in a dad joke sub

Edit 2: don't touch the thermostat

Do your worst!

How the hell am I suppose to know when it’s raining in Sweden?

Mathematical puns makes me number

Ants don’t even have the concept fathers, let alone a good dad joke. Keep r/ants out of my r/dadjokes.

But no, seriously. I understand rule 7 is great to have intelligent discussion, but sometimes it feels like 1 in 10 posts here is someone getting upset about the jokes on this sub. Let the mods deal with it, they regulate the sub.

We told her she can lean on us for support. Although, we are going to have to change her driver's license, her height is going down by a foot. I don't want to go too far out on a limb here but it better not be a hack job.

They were cooked in Greece.

43 year old white female, 5’2”, 107 lbs. 4+ years.

I am just looking for someone to guide me in the right direction by my blood test results. I have been battling autoimmune symptoms that are all over the place for over 4 years without a diagnosis. First doctor said GPA but she later changed it. I’m currently seeing my third rheumatologist and she’s wanting me to go to Mayo because in her opinion, I possibly have an autoimmune that doesn’t have a name yet or is very rare. Mayo is probably not going to be a possibility due to insurance and cost. My symptoms match the closest with MCTD but without going into a long story about my symptoms, I will just give my blood test results and ask for some type of information or point me in a direction that isn’t a well known disease or maybe in the research stage. I have done a ton of research on my own because the doctors have all admitted they have no clue what I have. I have averaged 2 to 3 blood test a year for the last 4 years. Each time I have tested positive for PR3 antibodies. The highest level was 4.5H and lowest was 2.3H. I have only tested positive for ANA once ever with an ANA pattern of Nuclear, dense fine speckled. My inflammation markers were really high when I was first tested but normal every other time since. My WBC, creatinine urine, protein total and a few other markers are low most of the time but right under the normal range, never extremely low so they don’t get looked into. I am tired of feeling like crap everyday and the fatigue and brain fog is causing me daily grief. I can’t be treated without a diagnosis and I’m on my fourth year. I don’t know where to go for help or what my next step should be other than keep seeing different rheumatologist until I get answers.

Hi!

First time posting here :)

So I'm trying to partially proteolyze a protein with Proteinase K and then using SDS PAGE. It's behaving really strangely though. I've tried different weight ratios of protein:ProK, and maybe I'm using too much proteinase K, but if I mix my protein with ProK and then IMMEDIATELY add loading dye with SDS/bromophenol blue/mercaptoethanol I still see just as much cleavage as I do when I let it sit for 10 min or 30 min at 37C. I find that really strange, since the company I bought it from said it can take 1-24 hours at a concentration of 50-100 ug/mL and I'm using less than that (4 ug/mL and a weight ratio of 1:50 (protein:PK)). What gives?