Do you guys know if vortexting or sonicating a solution can break antigen-antibody bonds?
I am basically capturing an antigen using beads that are coated with antibody. However, when I ran my experiment today, I did not detect any antigen on them. I spiked in antigen so I know it should have been there initially. Do you think vortexting/sonicating the solution screwed up the bonds? I can't find any info online.
Thanks in advance!
Logan Rivera
me no smart me have big muscle
Adam Howard
shoot wrong board. sorry about that. I frequent this the most whoops
Ryan Howard
It's okay, we can turn this in a manlet thread
Asher Gomez
fucking hate manlets
Ethan Hughes
short answer: probably not
long answer: it'd depend on the strength of the Ag/Ab interaction, which is a question of the immunoglobulin binding site. Iirc (bear with me I haven't done immunology in four years) the two primarily interact via H-binding which is fairly strong. I'd be surprised if vortexing can disrupt ligand binding
Daniel Davis
Everbody does
Nicholas Gutierrez
They are the worst
Andrew Rivera
Not on the immunology side but what were your controls? Do you guys run any basic igg stuff on the side to make sure the experimental procedure runs ok? The vortexing likely wouldn't but sonication is always a bugger.
Controls keep the science good and stop lab chad stealing your data.
Jaxson Torres
fuck brehs. Spent $800 on this shiet to replicate results from a paper and can't replicate the first step.
Why is scientific publishing so flawed? I try to repeat protocols and contact authors and most ignore or answer in some vague way. I've come to realize that even Nature papers are BS... starting to debate whether my PhD is worth is with all this faux science articles.
Some straight up report things that might have worked once out of many trials and never describe their failures; so pathetic and frustrating...
>tfw try to make mental gains and entire field is filled with con artists
Cooper Adams
I would say yes. I don't know about vortexing but sonication I would say almost definitely. Antigen and Antibody binding is usually just weak shit like Van der Waals or Hydrogen bonds. I know it can fuck up secondary-quarternary protein structure so I assume this would be similar. However it won't actually denature the antibodies themselves (covalent bonds are 10 years of daily SS on roids in comparison).
Sorry for offtopic mods, hope I answered your question nerd.
Adrian Lewis
I'm doing elisa using beads that capture an antigen and then using a detection enzyme that is labeled with HRP to look for the antigen.
When I have no antigen, I should not be detecting any fluorescence or should get minimal from background.
However my positive control which has the antigen has the same signal as my negative which indicates that either my beads aren't capturing the antigen or the secondary is not sticking to the primary-antigen complex.
i followed the published protocol from another paper exactly and couldn't see the positive/negative separation.
I only vortexed since my beads started to aggregate but I didn't think this could destroy any antigen-antibody bonds.
Jeremiah Rogers
it's possible...try running the experiment again without vortexing it
science is finnicky like that breh. It's taken me a year and a half to replicate results for my master's
Brayden Wood
I would say sonicating would be your best approach. Definitely better than vortexing anyway. I don't do any molecular stuff anymore but my beads would definitely stay attached to my target protein when I vortexed/centrifuged
Kevin Wilson
>I don't do any molecular stuff anymore but my beads would definitely stay attached to my target protein when I vortexed/centrifuged
I dont understand what you said. Does that imply sonicating disrupts the bond? why would sonicating be the best approach to not disrupt bonds; then why are you using vortexing/centrifuge
Jayden Hill
...
Camden Ross
If either are strong enough to produce cavitation, yes. Not that it would happen. Not that is would necessarily matter either depending on which bit your antibodies are detecting.
Are you sure you understand what you're doing tho? It sounds like affinity chromatography (done bad) followed by ELISA?
Eli Gray
cringe
Jacob Ortiz
Yup I'm just replicating this process (shown in image) which is well known. Conjugating and all this makes sense and there are a ton of papers on it.
I just can't get the full immunocomplex on my system working but I just started last week.
Can't figure out if it is the primary antibody coupling to bead issue, or antigen not binding to primary issue, or secondary detecting the primary-antigen complex issue. I'm following a kit with the same exact reagents from a paper but got shit results.
I really hate optimizing and going through step by step but that is science I guess... the only thing I didn't know was how much of an impact vortexting/sonicating has on breaking these bonds for the immunocomplex
Daniel Gray
While this thread is up, I'm trying to find a possible DNA-binding protein in anthracis. I ran an EMSA that suggests a new DNA-binding protein and I've used affinity chromatography and ran SDS-PAGE and get a strong band, but when I sent it out for N-terminal sequencing they found like 200 proteins in that band. I can eliminate things like RNA pol, but I have no idea how to narrow down what that protein may be. Or I'm full of shit and am wasting my time and don't know how to do anything.
Gabriel Richardson
Good, Veeky Forums is here. Can u help me with my orgchem hw? In return I'll lift heavy objects for u
Levi Thompson
Kekmao
Camden Green
Neither of those things will do shit.
What is way more likely to be the isssue (although I am not that familiar with this process) is the solution i.e. shit like Ph and ions and such. That can change the protein conformation (so the antibodies won't detect it cuz it's the wrong shape now) and the tendency of the primary antibody to like that to the bead.
Also chromatography is (usually) slow af.
Beyond that how have you got your antibodies is that the fucked up bit etc
Ryder Sanders
I just bought a primary anti-PSA that detects a PSA protein and the detection molecule that is biotinylated.
All three are from the same company so they should bind in sequence and should detect fairly well... I hope I didn't screw up there.
Easton Sanders
There are a million next steps you could do, the cheapest would be doing SDS-PAGE again. You can either draw the blot out or run it from scratch on a gel with better resolution at that point. There's also a bunch of Mass Spectrometry techniques (if you can afford them) or chromatography or both or neither.
Jose Mitchell
I don't know much about using a sonicator, but I do have two years of research with immunoassays, including immunoprecipitation which sounds like what you're trying to achieve.
We use SDS to separate our bead/Ab/antigen complex. This is usually enough to give us distinct bands at each molecule's molecular weight in western blot and PAGE.
Adrian Sullivan
Kek
Isaac Lee
>Antigen and Antibody binding is usually just weak shit like Van der Waals or Hydrogen bonds This is completely wrong, antibody/antigen complexes have one of the strongest binding affinities in nature, eclipsed only by biotin and avidin.
Jacob Ortiz
>orgchem >not Ochem
kill yourself so phat my dude!
Jaxson Nguyen
Curious why you're using beads for Elisa? Everytime I've run Elisa I always use capture plates (from nunc) and it always seems to work.
Ryder Bell
Sonicators do about as much as tapping a beaker on a desk (think when the qt barista is getting your foam just right, sometimes they tap to bring up the large bubbles, then swirl to mix back in the small ones). I'm being a little facetious but they don't usually do that much. They can raise temperature tho which might be of concern to OP.
Good advice too.
I don't know PSA well but if it's a serine protease it could digest itself and that could seriously fuck shit up. But I just don't know!
I feel I have to stick up for Van Der Waals and hydrogen now. They're often very strong too en masse.
Juan Collins
There might be something wrong with your column?
Evan Carter
>Ochem >not Orgo
Pleb
Elijah Cox
How heavy?
James Rogers
Never mind, I got it. Thanks bro
Brody Sanders
kek
Kayden Smith
I'm doing a microfluidic assay so running on a plate where it is immobilized and static won't work.
I'm basically going to capture antigen from plasma in a microfluidic device that will then detect fluorescent output on the beads - think of it as a mini flow cytometer. which is why I need a bead based assay rather than a well plate
Anyone know if it is easy to functionalize antibody onto a well plate? and is it straightforward
Anthony Brown
field specific jargon man is here
Hudson Jenkins
>sonicators do about as much as tapping a beaker on a desk >he doesn't ultrasonicate low energy plebian
Xavier Brooks
Kek, that's nothing m8, if you think modern scientific publishing is bad just wait until you see how much worse papers written thirty years ago are
Matthew Green
>easy to functionalize antibody onto a well plate I'm a biochemist so I don't even know what the word functionalize means, but you can get protein A coated 96 well plates which bind IgG
David Moore
Sweet that's exactly the type of stuff i was looking for.
So I can couple my antibody to the protein A coated wells and then run ELISA on it?
Man Veeky Forums gotta say thanks for helping so much. >tfw Veeky Forums helps out with my PhD work than a lot of resources at my university... who would have guessed
Justin Reyes
Yeah, I mean that's pretty much all that most elisas are unless you want to get fancy and do a sandwich method. I don't really know anything about microfluidics so I can't comment on your bead application
Ayden Parker
rewriting my deleted post cause I had it backwards, in this case you're doing a sandwich assay, where antibody linked to the plate picks up the antigen, then a second, detectable antibody is put on top then washed off so it will only stay if there is antigen there to keep it there during the wash, then you measure the detectable one by whatever method (fluorescence or chemiluminesence or whatever). There are more steps like blocking that are important so ask the dudes that you're buying from for a protocol, they normally have tech help lines with actual scientists that will come up with stuff for you
Andrew Thomas
Not OP, isn't that just a capture ELISA with a redundant first step? It's been a couple years since I ran these but I remember the order being protein sample, primary Ab, secondary Ab with HpOx, then whatever luminescent kit you have.
To me, adding your primary Ab first seems like a redundant first step, but maybe not.
Brayden Martinez
Get streptavidan coated beads with avadin labeled primary ab. Incubate that shit and they will bind. Then carry out the rest of the Elisa.
Adam Hernandez
Chemfag here, kindly fuck off with your """"science"""" and """"labwork""""
Camden Murphy
tfw there's better answers in this thread than this one :') >>Veeky Forums8817874
Wyatt Price
Don't you have some sort of protocol for your beads? (PROTIP: You do).
Try following instructions. If you spiked in antigen and you still didn't get a response then it's apparent that either 1) your reagents are fucked or 2) your procedure is fucked or 3) You're fucking retarded because both of them are fucked.
Why not just use an ELISA for detection or a HIS-tag for elution anyway?
Jace Martin
What if you did mass spec? You could get all 200 amino acid sequences and see if there's anything that's not on uniprot. You could see if the DNA sequence of an AA of interest is in a logical place within the genome of the bacteria.
You could go a step further and show via immunofluoresence that the protein is close by to DAPI-stained DNA.
Christian Wright
sorry about modern medicine you can abstain from it if you like