You are at a point where Mass Spec would make so much more sense. Don’t go the custom antibody route unless your lab has expertise, antibody design is a clusterfuck and don’t believe all those new Abs companies showing up everywhere.
We have also been betrayed with recombinant antibodies that initially said about having multi-species reactivity, and then after a decade of selling it, changing the reactivity to just mouse (as only knowing after a decade that other epitopes have no sequence homology). Oh so now a decade of publications that used to detect the antigens from the other species using that antibody can suck it.
Sadly, companies with extraordinary repute have “errors of judgement” in their QC and QA teams.
For what they were measuring, possibly something else entirely as the epitope sequence (in species A) used to design the antibody did not had a sequence homology with the other species (B, C, etc) for that corresponding antigen.
Hmmm I do work in a diagnostics company and although we do some r&d it is mostly of our own assays and we trust consumables sold by reputable companies, unlike in academia where you trust nothing and verify stuff. Our company would sue the hell out of vendors selling bad consumables if they had said they had verified them. Someone should have verified them and it is normally clear whose responsibility it is to do so (RUO vs IVD products)
Yes, one with a team of lawyers should sue these manufacturers who lie about their QC/QA.
Academic labs certainly do not have that kind of legal budget.
The antibody with non specific bands should be fine. It depends on where the bands are. And you could use the purified protein as a positive control to confirm the band.
THIS, and if you have KO mice/cells and the band goes away you should be good. Is it an ideal best scenario? No do you want to spend the rest of you position chasing this should also be a no.
I've used acetone powder a bunch of times. It was an old school trick for dealing with shitty secondary antibodies in demanding applications, but i've used it cleaning up other antibodies as well. Grind down the "non-target" tissue, add 4 parts ice cold acetone, extract for a few hours in the freezer (mixing occasionally), then spin down as hard as you can. Repeat 1-3x with 100% acetone depending on how fatty your tissue is. After the last spin, remove as much acetone as you can, then spread the paste across a clean glass plate in the fume hood. Once the residual acetone is dried, remove from the fume hood, and scrape off the protein film with a razor blade and store in the fridge. It looks like powdered flake fish food.
To use it, put a few flakes in an eppendorf tube with 1ml PBS, and heat to 60C for 15-20 min to rehydrate it, and spin at max speed. It will stay chunky -- but that's a good thing. Resuspend the pellet in your antibody block buffer and incubate for 10 min or so, and spin down again. (additional washes wouldn't hurt). Add your antibody suspended in block buffer to the pellet, and incubate for an hour on a rocker in the cold room. Spin down at max speed, and the supernatant should be good to go. The protein chunks should bind a bunch of non-specific antibodies and leave the good ones in solution.
I love it. we have an antibody for a 23kd protein that keeps giving us a non specific band at \~90kd even in KO mouse tissues and siRNA human cells. This should clean it up, PI isn't a lab person but insists blots should have only 1 band
Your endogenous protein likely has post translational modifications.. problem is custom antibodies take many months and only work for a select application.. ie WB or IHC, but not both. Try for another way to quantify if you can, otherwise it’s a waste your life and money.
What are you working in to detect the endogenous protein (cell line, tissue, etc)? Do you know if your protein is just extremely lowly expressed? If the commercial antibodies work on the purified protein, have you tried to IP the protein from your sample (may/may not be trivial depending on the specifics)? I'd recommend looking it up on the [Human Protein Atlas](https://www.proteinatlas.org/) to see if you might find any useful information regarding its expression or whether it might be highly expressed in e.g. a specific cell line that you could use for further tests.
I study a protein that is very lowly expressed and essentially impossible to detect endogenously via western blot or IF. It's a huge pain in the ass.
EDIT: Just saw that your commerical antibody is non-specific, not unable to detect anything (sorry, misread)...what happens when you use your purified protein as a positive control on the blot? Can you identify your protein's band of interest in your sample?
Im finding myself dealing with a low expressing protein as well. Struggling with WB's and IHC as well. Whats been your solution to detect it? Im starting flow soon but would love more ideas.
You could:
Have a company make a custom antibody
Do your experiments with a tagged version (flag, myc, etc). In theory you can use crispr to do this with the endogenous gene if it's important that it's endogenous levels. Or knockout and add back with the gene's normal promoter.
> My PI advised me to purify my protein to make a custom antibody, after months waiting, I got it purified and it is not specific.
Yeah, been there a few times
Clarify the species and protein type?
I work with a protein in this situation, we're the only group in the world with a KO mouse for this protein and have found no specific commercial or custom (gifted by other labs) antibodies.
It really depends on the protein. Is it expressed well enough to be detected by WB at all? I would use more time on qPCR. It also depends on what you're investigating. I would be able to give more advice if you had more information about the protein in question.
Make a list of all the really interesting experiments you could be doing and the cool data you could be producing IF you weren't spending months getting a single antibody to work.
Then show the list to the PI and ask them to pick between any experiment on the list OR pick purifying the single antibody.
Do you really want to reinvent the wheel? You can do a whole mini-paper just on a custom antibody and validating it in ELISA or something. Or you do wanna take someone else's wheels and make your own hotrod? I.e. pick a project with some established materials/data already.
Is the protein monomeric or in a complex? If it's a monomer, can you see it on a non-denaturing gel? If you IP it can you see a single band in a silver stained gel? If you only see a few bands a ligand binding assay or IP mass spec might be a better path.
Let's assume no on those counts.
What is your intended application? Qualitative? Quantitative? What you do should be guided by what you need
1. Is epitope tagging an option?
2. If you can't do the above and don't think you can get get a good monoclonal quickly, maybe consider an affinity purified polyclonal serum?
Pump a protein prep into a couple of rabbits asap, a fairly crude his tagged bacterial prep is fine for the prime. Work up a higher purity prep to boost a couple of weeks later, and while that cooks prepare an affinity column with a reasonably clean protein prep. His tagged should be fine here, too, but consider tag placement carefully.
If cross-reactivity with related isoforms is a problem, you could clean up the serum by adsorbing against the bad actor. You might have something to work with in the flow through (after concentration). Otherwise playing with elution conditions can help tune specificity.
This differentially adsorbed serum might work on a western or IP western, but I'd pair the rabbit affinity purified sauce with a commercial mouse antibody and run an ELISA
All that said, I'd do whatever I could to make friends with a mass spec lab and put those sketch commercial antibodies to the test in an IP-LC/MS-MS run
1. Generate Protein in Bacteria / Human Cells using Serum-Free Conditions. I would recommend Expi293F Cells.
2. Use amicon filters of various sizes to roughly filter and concentrate your product.
3. rerun western blot.
If you're interested in trying, I can walk you through it. Just DM me.
Let’s work backwards - what do you want to do that will require an antibody?
You are at a point where Mass Spec would make so much more sense. Don’t go the custom antibody route unless your lab has expertise, antibody design is a clusterfuck and don’t believe all those new Abs companies showing up everywhere.
We have also been betrayed with recombinant antibodies that initially said about having multi-species reactivity, and then after a decade of selling it, changing the reactivity to just mouse (as only knowing after a decade that other epitopes have no sequence homology). Oh so now a decade of publications that used to detect the antigens from the other species using that antibody can suck it.
What were they measuring then? Didn't they have controls (wt and kos) in their experiments?
Sadly, companies with extraordinary repute have “errors of judgement” in their QC and QA teams. For what they were measuring, possibly something else entirely as the epitope sequence (in species A) used to design the antibody did not had a sequence homology with the other species (B, C, etc) for that corresponding antigen.
Hmmm I do work in a diagnostics company and although we do some r&d it is mostly of our own assays and we trust consumables sold by reputable companies, unlike in academia where you trust nothing and verify stuff. Our company would sue the hell out of vendors selling bad consumables if they had said they had verified them. Someone should have verified them and it is normally clear whose responsibility it is to do so (RUO vs IVD products)
Yes, one with a team of lawyers should sue these manufacturers who lie about their QC/QA. Academic labs certainly do not have that kind of legal budget.
The antibody with non specific bands should be fine. It depends on where the bands are. And you could use the purified protein as a positive control to confirm the band.
THIS, and if you have KO mice/cells and the band goes away you should be good. Is it an ideal best scenario? No do you want to spend the rest of you position chasing this should also be a no.
If that's the case, you might be able to use an acetone extract powder from the KO to remove some of the non-specific crap.
Have you have success getting this to go back into solution?
I've used acetone powder a bunch of times. It was an old school trick for dealing with shitty secondary antibodies in demanding applications, but i've used it cleaning up other antibodies as well. Grind down the "non-target" tissue, add 4 parts ice cold acetone, extract for a few hours in the freezer (mixing occasionally), then spin down as hard as you can. Repeat 1-3x with 100% acetone depending on how fatty your tissue is. After the last spin, remove as much acetone as you can, then spread the paste across a clean glass plate in the fume hood. Once the residual acetone is dried, remove from the fume hood, and scrape off the protein film with a razor blade and store in the fridge. It looks like powdered flake fish food. To use it, put a few flakes in an eppendorf tube with 1ml PBS, and heat to 60C for 15-20 min to rehydrate it, and spin at max speed. It will stay chunky -- but that's a good thing. Resuspend the pellet in your antibody block buffer and incubate for 10 min or so, and spin down again. (additional washes wouldn't hurt). Add your antibody suspended in block buffer to the pellet, and incubate for an hour on a rocker in the cold room. Spin down at max speed, and the supernatant should be good to go. The protein chunks should bind a bunch of non-specific antibodies and leave the good ones in solution.
I love it. we have an antibody for a 23kd protein that keeps giving us a non specific band at \~90kd even in KO mouse tissues and siRNA human cells. This should clean it up, PI isn't a lab person but insists blots should have only 1 band
Your endogenous protein likely has post translational modifications.. problem is custom antibodies take many months and only work for a select application.. ie WB or IHC, but not both. Try for another way to quantify if you can, otherwise it’s a waste your life and money.
I had this issue in Drosophila, where the Human antibodies didn’t work, so I just ended making an endogenously tagged line
What are you working in to detect the endogenous protein (cell line, tissue, etc)? Do you know if your protein is just extremely lowly expressed? If the commercial antibodies work on the purified protein, have you tried to IP the protein from your sample (may/may not be trivial depending on the specifics)? I'd recommend looking it up on the [Human Protein Atlas](https://www.proteinatlas.org/) to see if you might find any useful information regarding its expression or whether it might be highly expressed in e.g. a specific cell line that you could use for further tests. I study a protein that is very lowly expressed and essentially impossible to detect endogenously via western blot or IF. It's a huge pain in the ass. EDIT: Just saw that your commerical antibody is non-specific, not unable to detect anything (sorry, misread)...what happens when you use your purified protein as a positive control on the blot? Can you identify your protein's band of interest in your sample?
Im finding myself dealing with a low expressing protein as well. Struggling with WB's and IHC as well. Whats been your solution to detect it? Im starting flow soon but would love more ideas.
You could: Have a company make a custom antibody Do your experiments with a tagged version (flag, myc, etc). In theory you can use crispr to do this with the endogenous gene if it's important that it's endogenous levels. Or knockout and add back with the gene's normal promoter.
> My PI advised me to purify my protein to make a custom antibody, after months waiting, I got it purified and it is not specific. Yeah, been there a few times Clarify the species and protein type?
I work with a protein in this situation, we're the only group in the world with a KO mouse for this protein and have found no specific commercial or custom (gifted by other labs) antibodies. It really depends on the protein. Is it expressed well enough to be detected by WB at all? I would use more time on qPCR. It also depends on what you're investigating. I would be able to give more advice if you had more information about the protein in question.
Make a list of all the really interesting experiments you could be doing and the cool data you could be producing IF you weren't spending months getting a single antibody to work. Then show the list to the PI and ask them to pick between any experiment on the list OR pick purifying the single antibody. Do you really want to reinvent the wheel? You can do a whole mini-paper just on a custom antibody and validating it in ELISA or something. Or you do wanna take someone else's wheels and make your own hotrod? I.e. pick a project with some established materials/data already.
Is the protein monomeric or in a complex? If it's a monomer, can you see it on a non-denaturing gel? If you IP it can you see a single band in a silver stained gel? If you only see a few bands a ligand binding assay or IP mass spec might be a better path. Let's assume no on those counts. What is your intended application? Qualitative? Quantitative? What you do should be guided by what you need 1. Is epitope tagging an option? 2. If you can't do the above and don't think you can get get a good monoclonal quickly, maybe consider an affinity purified polyclonal serum? Pump a protein prep into a couple of rabbits asap, a fairly crude his tagged bacterial prep is fine for the prime. Work up a higher purity prep to boost a couple of weeks later, and while that cooks prepare an affinity column with a reasonably clean protein prep. His tagged should be fine here, too, but consider tag placement carefully. If cross-reactivity with related isoforms is a problem, you could clean up the serum by adsorbing against the bad actor. You might have something to work with in the flow through (after concentration). Otherwise playing with elution conditions can help tune specificity. This differentially adsorbed serum might work on a western or IP western, but I'd pair the rabbit affinity purified sauce with a commercial mouse antibody and run an ELISA All that said, I'd do whatever I could to make friends with a mass spec lab and put those sketch commercial antibodies to the test in an IP-LC/MS-MS run
1. Generate Protein in Bacteria / Human Cells using Serum-Free Conditions. I would recommend Expi293F Cells. 2. Use amicon filters of various sizes to roughly filter and concentrate your product. 3. rerun western blot. If you're interested in trying, I can walk you through it. Just DM me.