BIOCHEM RESEARCH HELP

[Anonymous No.10892947]

I retrieved a sample of fish skin mucus and I will be trying to purify and characterize some proteins from it.
As my professors recommended me I didn't add anything to the sample and I'm storing it in a fridge (4 C°).
But I read on a paper that proteins will last only 1 month with that method.
I'm not concerned for an amicrobial attack since it's mucus and has antimicrobic activity, I'm worried that proteins will degrade and I won't be able to characterize them anymore...
Should I freeze it? Or will it be fine? Considering 16 days has passed already, uni and profs are unavailable until september and I still haven't designed the experiments.

[Anonymous No.10892952]

Here's the paper, but it's about purified proteins and their activity span, I haven't found anything about skin mucus storing, it's surely different

[Anonymous No.10892954]

you could add some protease inhibitor

[Anonymous No.10892973]

How hard is it to get a new sample?

[Anonymous No.10892996]

>>10892973
Hard enough, I had to personally search and spearfish two specimen of the specie I was looking for so I could extract fresh mucus, which obviosuly isn't available in a fish shop.

[Anonymous No.10892999]

1) how much sample did you get?

2) why put it in the freezer? you're concerned with proteins, store that shit in -20°C or -80°C freezer.

3) >>10892954 is a start. Look into anything general to stabilize proteins, however, if you do 2, you shouldn't need 3 until you're ready to purify / characterize.

4) Which types or proteins are you looking to purify / characterize? Because there's a shit ton of different types and you're sure as hell ain't gonna get the one's you want if you don't have enough sample.

[Anonymous No.10893001]

>>10892947
>>10892952
>>10892996
Making a lube product for us OP?

[Anonymous No.10893034]

>>10892999
>1) how much sample did you get?
around 4 mL, but there's a bit of sea water, and the mucus precipitated, and the precipitate it's around 1,5-2 mL (don't have the sample at hand rn)
>2) why put it in the freezer? you're concerned with proteins, store that shit in -20°C or -80°C freezer.
Yeah that's what I meant
>3) >>10892954 is a start. Look into anything general to stabilize proteins, however, if you do 2, you shouldn't need 3 until you're ready to purify / characterize.
Alright so you think protease hinibs will be enough for let's say a couple months?
>4) Which types or proteins are you looking to purify / characterize? Because there's a shit ton of different types and you're sure as hell ain't gonna get the one's you want if you don't have enough sample.
It should be enough imo, since I will isolate them via affinity chromatography. That's why I need them to maintain their chemical activity.
>>10893001
N-no

[Anonymous No.10893065]

>>10893034
Man, I don't think you understand how little protein you'll get. Either you're getting some extremely ubiquitous, or you should just use a transgenic approach and express your protein in E. coli or something, because purifying barely a few micrograms is going to feel like dogshit.
And even then, your affinity chromatography might still pick up all kinds of clutter, even if your protocol for protein extraction is decent.

[Anonymous No.10893110]

>>10893065
But I don't need to do anything else other than characterizing it, it's a new protein so I just need to do that (for now). Why wouldn't a small amount of purified proteins be enough for that?
As I said this protein isn't characterized yet so I can't do anything transgenic without knowing the aa sequence.
Generally what's the amount of purified proteins normally required for a protein characterization precedure?

[Anonymous No.10893122]

Depends on what kind of characterization you want..

What type of characterization are you doing?
Biochemical properties?
Actual structure? You want a crystal?
You're deducing the AA sequence from it?

How do you know what the protein is without knowing what it is? How do you know it's a new protein?

[Anonymous No.10893211]

>>10893122
AA sequence and molecular weight should me enough for now, I don't really need the tridimentional structure.
It's biological activity should be easy to visualize, 25 microliter of skin mucus react in a solution with the protein'l substrate in a similar reaserch.
This protein is known, just not the exact one in the specie I'm studying, it's present in other species which share the same order with my specie but differ (the same type of protein from 2 species of that subirder has been characterized and they are different, so I presume mine will be too since they are phylogenetically distant enough)

[Anonymous No.10893250]

>>10893211
So you have the range of MW you can look for.
Some kind of extraction protocol and 2D page + LC-MC could do it.

It's still a very dubious way of doing molecular research as you have no clue what your protein actually does and you have a complex environment will all the clutter the fish intrinsically brings along (glycosylation patterns, mucus, sugars, all the different proteins).

Why not just sequence its genome, find the relevant DNA stretch with BLASTing against the homologues and then trying to make the protein yourself?

[Anonymous No.10893512]

>>10893034
>hinibs
nice troll asshole, hope you choke on a fish bone one day

[Anonymous No.10893556]

>>10893250
I know the biomolecules it reacts with, i think it's doable, why do you think it's weird?
I mean, i have at least 3 papers that did basically the same but with different species, I'm not doing anything new, I'm playing it safe also because I'm just and undergrad.
I don't think my uni would let me do Blast and all that fancy stuff with DNA, I think it's too expensive.

If you have any other tips I'm all ears, the professor I'm doing this with isn't even a biochemist so I'll need all the help I can get

[Anonymous No.10893565]

>>10893556
>I don't think my uni would let me do Blast and all that fancy stuff with DNA, I think it's too expensive.
bruh if you don't at least try they'll think you lack confidence and you're a little baby good luck getting a PhD with that attitude

[Anonymous No.10893568]

>>10893556
>biomolecules

[Anonymous No.10893569]

>>10893001
All natural, babyyy

[Anonymous No.10893578]

>>10893556
>I don't think my uni would let me do Blast and all that fancy stuff with DNA, I think it's too expensive.
Ok but now you're too obvious, it's not funny, fuck off

[Anonymous No.10893592]

>>10893556
Let's say you have the biomolecules it reacts with.
Let's say you can purify it by letting your protein you're after interact with said biomolecules.
How do you know for sure you found your protein?
A complex environment like fish mucous membrane is full of proteins of all kinds, it's like finding a needle in a haystack. And even when you have the biomolecules it interacts with, you'll always have cross reaction from unwanted stuff.

If you have the protocls etc. I mean then go ahead. Just don't put it in the fridge...

[Anonymous No.10893609]

>>10893122
>>10893250
>>10893592
He's asking how to store it properly and whether he can still use it you dumb condescending asshole

[Anonymous No.10893624]

>>10893609
I told him at the start he should freeze it. ALWAYS freeze your biomolecules. Fridge is just a recipe for deterioration.
Then we started having a conversation by him answering my questions.
I'm pretty sure he's mature enough to fend for himself, and he did so too, so with all due respect, fuck off.

[Anonymous No.10893628]

>>10893624
go back to fucking reddit you dumb pseudo, freezing water is different from freezing glycerol, storage of protein is more complicated than you think. also nice formatting, did you catch that on reddit?

[Anonymous No.10893662]

>>10893628
I genuinely have no clue how to respond decently to this. You like having this hostile attitude?
Sure it's "more complicated" to use a cryoprotectant and know the reasons why, but that's not the point. The point is, freezing is better because you can fucking keep it quasi indefinitely. If you just keep it in the fridge, your sample is gonna get fucked in the ass if you don't do something with it reasonably quickly.

[Anonymous No.10893680]

>Sure it's "more complicated" to use a cryoprotectant and know the reasons why, but that's not the point. The point is, freezing is better because you can fucking keep it quasi indefinitely. If you just keep it in the fridge, your sample is gonna get fucked in the ass if you don't do something with it reasonably quickly.
No fucking shit. Are you telling me that if I am about to freeze something, it sure is smart to use a cryoprotectant? Woah.
Who even considers putting bio samples in a fucking fridge?
Did you ever put some food in a fridge, forgot about it and noticed how it began to smell like shit?

[Anonymous No.10893688]

>>10893680
If you could tell me what crawled up your ass, I might be able to help you, because dear lord, you're quite catty dude.

[Anonymous No.10893693]

>>10893688
a woman wrote this

[Anonymous No.10893698]

>>10893688
it is you girl who crawled up my ass and i liked it

[Anonymous No.10893701]

>>10893565
They don't give a shit, I'm already doing much more than what I'm supposed to do
>>10893568
https://en.m.wikipedia.org/wiki/Biomolecule
>>10893592
Yes I was intending to follow already working protocols, they use like 3 different chromatography columns subsequently to extract the protein, it's not as simple.
>>10893628
No need to get angry, if you disagree with something just explain why, I've read it's okay to freeze proteins without additives but with every further freezing the sample degrade (just like when you defrost meat you shoudnt freeze it again) so you need to use them after you defrost them.

So, I should freeze it.
But I can't do it before at at least 33 days from the sampling.
Will it be fucked? Oh God please no I can't get anymore of it I'm fucked if it gets degraded please nooooooooooooo

[Anonymous No.10893704]

>>10893693
Or, I just don't feel like dealing with aggressive shitstains over the internet because you feel like it.
Learn to be productive and vent your anger to somewhere else.
If you're frustrated, let's hear why, perhaps you and all of us can learn something from it.
If you're not about to share, and you don't feel like contributing to a thread, kindly fuck off.

[Anonymous No.10893707]

Just collect more fish mucus

[Anonymous No.10893709]

>>10893701
>let me show you my http of this word "biomolecule" which is real, since it is on wikipedia. checkmate non-scientists

[Anonymous No.10893711]

>>10893701
>they use like 3 different chromatography columns subsequently to extract the protein
Really bro, they should be teaching you how to do that shit in like year one lol. Which shithole do you go to uni at.

[Anonymous No.10893715]

>>10893704
why did i read this musically as a song from Pink

[Anonymous No.10893722]

>>10893707
The only good answer but OP probably left ages ago

[Anonymous No.10893735]

>>10893707
Can't, it's not generic fish mucus is fish mucus of a determined specie, the only thing possible to get more is to ask an aquarium but not many have this fish unfortunately. I can't go back where I fished the two either.
>>10893711
Not US, did almost no labs cuz we are too many. We briefly talked about lab methodics.
First time I do this shit. But I should be able to do it, doesn't seem too difficult.
>>10893722
Never left

[Anonymous No.10893749]

>>10893735
>Never left
Wait, you are the OP? fucking hell girl, if you want real help, ask on some other shithole but not here

[Anonymous No.10893866]

>>10893735
Obtain the mucus

[Anonymous No.10893966]

>>10893866
Gather the fluid

[Anonymous No.10894826]

>>10893966
Acquire the liquid

[Anonymous No.10895884]

>>10894826
Procure the secretion

[Anonymous No.10896952]

>>10893556
>If you have any other tips I'm all ears, the professor I'm doing this with isn't even a biochemist so I'll need all the help I can get
Do you know what the protein is? I'm assuming you do. Order cDNA insert, sub-clone into an expression vector and recombinently-express in E. Coli. To do any sort of biochemistry or biophysics or structural biology studies, you're going to need tons more than you'll get from fish mucus or whatever. Fortunately for you, we have recombinent DNA technology.

Your first challenge is going to be expressing and purifying the construct.

[Anonymous No.10897043]

>>10896952
I get it, but all of that will be possible only after I isolate and characterize my protein from the mucus since not even its primary structure is known, right? You are advising me to do the recombination after I isolate the protein, so that I have a higher amount of it for further analysis, right?
(Thanks anon)

[Anonymous No.10897179]

>>10896952
This is bollocks.

>>10893592
Someone's done a small amount of... I dunno ELISA or something and thinks they're a genius.

>>10893624
If it's already a problem of there being a limited amount cryoprotectant isn't necessarily the best idea.

>>10893701
Do you know why you're using three columns? I can't see a link to the paper.to check out the protocol. Is this undergrad bullshit? In which case worry less. By September it will have been approx a month.

[Anonymous No.10897347]

>>10897179
They use the first chromatography column to isolate the few proteins that react with the reagent, then a desalting column and finally a fast protein liquid chromatography column to separate the 2 similar proteins from each other.
That will be another factor I'll have to consider, I don't know how many proteins are in that mucus, could be one, could be 2 or 3...
It's for my undergrad thesis, but I'm doing all of this to possibly get published to be more employable n shit.

>By September it will have been approx a month.
Meaning my proteins will last? I'm trying to contact my prof to get it frozen, still waiting to get a reply...

What would you do man? I'd freeze it, but again, at - 20 or - 80 C°?

[Anonymous No.10897551]

50/50 with 50% glycerine

[Anonymous No.10897990]

>>10897043
>I get it, but all of that will be possible only after I isolate and characterize my protein from the mucus since not even its primary structure is known, right?
If the sequence of the protein is not known, then I would guess the protein itself isn't known. The first paper(s) that publishes the cloning of a gene of Protein X and the purification/characterization of Protein X would have this information. Do you know the name of the protein that you're working on?

>You are advising me to do the recombination after I isolate the protein, so that I have a higher amount of it for further analysis, right?
Not necessarily. My advice is that recombinent protein expression will be necessary to generate enough protein for "characterization" or any sort, whether you want to perform structure-function studies, binding partner characterization, biophysical studies, etc. The "isolation" process is what we refer to as "protein purification" or "prepping protein". Your protein construct would have an affinity tag, and you can purify protein via any number of chromatography steps (affinity, ion-exchange, size-exclusion).

What tag to use and what purification strategy, though, is a bridge to cross when we'll come to it. My question now is if you know that name of the protein you're working on, and if anyone has cloned the gene for this protein or purified this protein before.

[Anonymous No.10897995]

Continued from >>10897990

>>10897179
>This is bollocks.
What is bollocks about what I said? On face value, I said that if he wants to crystallize the protein or do biochemical or biophysical characterization (casting a wide net here), then he'll need to recombinently-express protein. He's not going to get enough pure protein to set crystal trays or whatever from fish mucus.

Now, if he doesn't know the name of the protein or what the protein is, then that's a whole different ballgame. This post >>10893250 talks about his best bet, optimize extraction protocol and submit for mass spec. Here is an technique that is used: https://www.ncbi.nlm.nih.gov/pubmed/20704860..

[Anonymous No.10898211]

>>10897995
>I said that if he wants to crystallize the protein
I see what you're saying now, I misread. A lot of people are doing cryoEM now in preference to crystallography, there's also a bunch of assumptions built in to some of the takeaways people make from protein crystallography, but what you say is valid. I can't see this being anything other than a shot gun proteomics thing or something very similar though, I assume it's going to be "look what the mass spec said about these fish, and look at the minor difference in our fish" or something to that effect.

>>10897990
>and if anyone has cloned the gene for this protein
I merely want to say that how you're wording things sometimes is problematic and that's part of why I was confused earlier, like this.

>>10897347
>Meaning my proteins will last?
It's more important in this case to follow the prior protocol, they are suggesting it will last for that period of time in a way that is still relevant to what they're doing in the paper. It says 1 month, you'll have had them for 1 month. It may not work and you'll get to discuss that. What scale are we at? If you can use a small amount of mucus is it worth doing some smaller scale chromatography like PAGE or even an agar gel to maybe get some data points for the mucus breaking down or whatever?

[Anonymous No.10898222]

>>10897990
No, as I said the type of protein and it's general function is known, but the protein in the specie I'm working on isn't, and it's higly probable it's different from the ones known. that's why I got a sample of mucus. I'm sorry I'm not being more specific but I don't want to dox myself and my research.
It hasn't been purified before nor its gene is known.

[Anonymous No.10898405]

>>10898211
OK first of all I found a paper which basically says lyophilizing fish mucus decreases its protein amount, while freezing it at - 20 or - 80 don't, or at least no as much as lyophilization.
So now I know better what to do.
Although the amount of mucus they gathered scares me, it's true that they run many tests but goddamn they got 30 ml of mucus...
Here's the paper, mind that the types of protein they analysed don't comprehend mine but the total amount factor is still useful
https://www.ncbi.nlm.nih.gov/m/pubmed/27697558/?i=16&from=mucus%20frozen

Now about SDS page, I have around 1,5 ml of mucus, surely enough for a page (right?), but it would be pointless to run it before the affinity chromatography, I mean there are surely hundreds of proteins in there, it would be proficient to run it after I isolate the type of protein I'm looking for to separate them.
I have 2 questions: how sensible SDS PAGE is? Can it separate proteins with similar molecular weight? How much different in weight proteins have to be to be separated?
And what's the minimal amount of purified proteins necessary to have a good gel to analyse?
Thanks in advance bro

[Anonymous No.10898410]

>>10895884
Collect the eject

[Anonymous No.10898692]

>>10898405
>I have 2 questions: how sensible SDS PAGE is? Can it separate proteins with similar molecular weight? How much different in weight proteins have to be to be separated?
You make or buy the gel of the appropriate pore size/concentration (same thing) for the size of protein you're looking for and then you run the thing until you get the band separation you want. The amounts of protein can certainly be a problem, but you can work with very small amounts with this, it's probably about the best process for small amounts when you're not sure what you're looking for. If you want to do this find someone who's done it before, one of the PAGE precursors is a neurotoxin if you're making it and there's a few things you need to be mindful of.
>And what's the minimal amount of purified proteins necessary to have a good gel to analyse?
That's more a question of how you're staining it/finding any bands. You can even keep the bands closer together and do 2d page or stamp out the gel and do a stage of chromatography. It might also be worth doing isoelectric focusing. That adds cost, but improves separation. You can do a lot with gels dude.

You probably do want to run the affinity column first as that's like the clean up stage, PAGE can have problems with dirty samples. I'll have a look at the paper at some point and see if anything else comes to mind. Also I'm guessing with that amount of mucus it's like the teeny tiny chromatography things in eppendorf tubes and what not.

[Anonymous No.10898701]

>>10898692
Yeah, get rid of all the ubiquitous stuff first, like Ubiquitin.

[Anonymous No.10898708]

>>10898692
>>10898405
Oh, okay, still worried about storage. Look, if you're really worried you can probably just freeze it as is, probably wise to aliquot in case you fuck up somehow with one. So long as you aren't freezing and thawing that same sample over and over it's probably not going to make a massive amount of difference. Also if the specific protein is a particularly windy one or something else that makes it resistant to enzymatic degradation, it might be that you're worrying over nothing.

[Anonymous No.10898734]

>>10898410
Drain the membrane.

[Anonymous No.10898902]

>>10893711
>They teach that shit in the first year
>They're teaching it in the second year
>Most people and teachers don't encourage us to do a paper

Fuck my life.

[Anonymous No.10899297]

>>10892947
try something, you can always get new fish

[Anonymous No.10899309]

>>10898692
OK got it. I'll have to look a bit more into Page and everything else.
>Also I'm guessing with that amount of mucus it's like the teeny tiny chromatography things in eppendorf tubes and what not.
About that... What will happen if the proteins aren't enough? Nothing will come out of the column? Maybe a couple droplets?
Should I add to the column some buffer of some kind that doesn't get filtered so that I can extract the proteins with it? That's a problem.
>>10898708
Yes it's the priority right now, I can think abou designing the experiments after I secure the sample.
If, for an unfortunate event, I didn't find a proper freezer in my uni, could I set a normal freezer to - 20C°? I know they normally are set at -18...

[Anonymous No.10899812]

>>10899297
I wish I could

[Anonymous No.10900964]

Pls someone, my prof hasn't been fucking answering the phone for 4 days what do I do is my home freezer an option

[Anonymous No.10901064]

>>10900964
if ur home freezer doesn't hit -20/-80 i'd say no bud

[Anonymous No.10902148]

>>10901064
I fucking hate boomers omfgggg

[Anonymous No.10902279]

>>10902148
Can you tinker together some liquid nitrogen apparatus

[Anonymous No.10902287]

>>10900964
I hate women so much

[Anonymous No.10903823]

>>10902279
I emailed my biochem prof... Hopefully he can help me
>>10902287
He's a him