I'm performing IPs for the purpose of sending the eluates to mass spectrometry to determine the protein-protein interaction network of my protein of interest. I typically elute the proteins off the beads with 300ul 0.5M ammonium hydroxide and then use a SpeedVac to lyophilize the proteins/evaporate the NH4OH. This evaporation process often takes >4 hours. I set the SpeedVac to 30C. My question is, would a higher temperature make this process quicker? Or would I risk damaging the proteins? Or would that not matter since the proteins will be digested/fragment for mass spec anyway?
I’m finishing my clinical right now and i’m in blood bank. Our SOP says to do an elution when a DAT is compliment positive and IgG negative. I just dont understand the reasoning, and the techs at the hospital can’t explain it.
If anyone knows why an elution is performed i’d love to know
Thanks!
Our lab is currently validating a Kingfisher Flex with the aim of moving to it using the MagMAX Microbiome Ultra Nucleic Acid Isolation kit for use on concentrated wastewater samples, but we’re running into problems. When we take the elution plate for PCR we’re finding that a lot of the elution buffer is missing (should be 50uL but the most we’re getting out of any well is 35). I’ve just checked the pipette we used to dispense it and it’s behaving perfectly, so the only thing I can think of is either that the kingfisher itself is getting too hot and the buffer solution is evaporating, or we’re losing some when the beads are removed. Has anyone else run into a similar problem and if so how did you fix it? Thermofisher are being very unhelpful.
Is CTAB buffer light-sensitive? Can I keep it at room temperature in a clear glass bottle?
Same question for elution buffer composed of Tris-HCl (pH 8.5)
I don’t think anything would happen because the ions would just pass through the column… but clarification is appreciated
I am trying to understand IEX gradient elution a bit more. If you have a buffer A with no salt and a buffer B with 1 M NaCl and you’re at 50% pump A, 50% B, is it fair to assume that you’re now at 0.5 M NaCl? Is it that simple or are there other equations to calculate this?
Hello everyone,
I would like to officially introduce myself and my company to this sub. I have been a long time lurker and somewhat active member of r/hemp and r/cbg and have recently been interested in r/hempflowers. My name is Gian and I operate a company called Elution. We have been in the hemp/CBD space for 2 years trying our hands at farming, cloning, and more recently, retail products and extraction. Our team has a great background for plant cultivation (Me- B.S. in Horticulture) (Jason - PhD in Botany) (Gavin - Masters in Botany) and we recently discovered we can grow some pretty darn good smokable hemp flower.
After working closely with RuBi Hemp Co. last season, producing over 100k clones, and pheno hunting for some excellent smokable flower varieties, we are extremely excited to release our smokable flower and other CBD products to the public. Our favorite two varieties thus far are Cantaloupe Crush (yes it actually does smell like cantaloupe) and H5 CBG, but everything we carry is great and smelly. We painstakingly worked to grow, harvest, dry, trim, and slow cure our flower over the past few months to get it ready.
In addition to our CBD and CBG varieties of smokable flower we also have two different types of hemp cigarettes. These being a pure CBG cigarette and a CBD/CBG blended cigarette. We were really excited to make these because they allow us to produce a Total THC compliant pre-rolled flower product that only utilizes flower. Due to the really low THC, and less sticky nature of CBG we don't have to use any fillers like trim or biomass in our cigarettes to get a good consistancy or low THC blend. Super exciting!
We are dedicated to producing high quality products and running an ethical business that is liberal with information and transparancy so please don't hesitate to reach out to me or us with any questions. I'd love to hear what everyone thinks about CBG in particular and if you have noticed any benneficial effects from it as I have. Anyway, thanks for reading my long post and happy holidays!
- Gian
Hey everyone,
I am a relatively recent chem grad who is getting his feet wet in the analytical sector of the pharmaceutical industry. Most of the characterization/separation techniques we employ (GC, Titronics, etc.) I have a decent theoretical grasp on. However, I am still trying to figure out how to analyze molecules of interest in RP-HPLC and predict elution orders to a relative level of certainty when employing gradient-based elution. I understand that each separation is different (stationary phase packing, mobile phase, etc.), but is there a general rule of thumb in gradient elution for RT order (especially for small molecules [MW < 1000g/mol])? Is it based primarily on polarity, molecular size, stereochemistry, or what? Isocratic separations seem to be much more straight forward. If there are any resources anyone can recommend, I will gladly read them. I haven't found many resources in my search that have answered my questions.
Thanks!
Hello,
I have a question, out of curiosity: would it be possible to elute a his-tagged protein from a NiNTA column with pyrazole instead of imidazole?
Thanks for your opinions!
why do we need a large amount of NaCl in order to elute an acid in anion exchange column chromatography?
So I was talking with a coworker about this the other day and he completely didn't relate to this, but I sometimes find that at the end of the day, after I've spent say, 6 hours doing washes on a solid phase extraction plate full of patient samples (I work in a toxicology lab and we're small, so I do a lot of stuff by hand) and it's finally time to elute in our well plate I will suddenly picture in my head knocking over the bottle of elution solvent and completely soaking the entire plate of columns and with WAY too much solvent and losing all 96 wells and having to start over. It's not a pleasant thought, but I can't seem to help it -- almost like when you're standing next to a cliff and you picture throwing yourself off even though you'd never actually want to. Does that make sense? Do other people do that or am I crazy?
So my group wanted to separate acetaminophen, caffeine and aspirin using a reversed phase hplc-uv. So theoretically, caffeine the most polar will elute first followed by acetaminophen. However, the chromatogram shows that caffeine has a longer retention time than acetaminophen?? can anyone help explain why
You know the deal, I'm most kits out there is a small bottle of EB used to collect your precious material off a column. Our lenti core requires you to elute in water. I have not given it much thought but I figured I would poll the audience and see if using water over eb hurt or help yields and downstream processes.
Hi guys, I'm wondering if anyone does multiple elutions with the RNeasy Micro kit from Qiagen. I feel like there is probably more RNA on the spin column membrane so I was planning on eluting multiple times into different collection tubes (or the same one?). Anyone have any thoughts or experiences?
Hi, I started looking at protein purification today and didn't understand the elution step in affinity chromatography. Say our binding ligand on the beads is glucose (that traps the desired protein), why are we adding more glucose to compete for the binding to our protein? I don't get how that will dislodge the proteins, shouldn't we put something that competes for the ligand even more than the desired protein? Unless if by adding free glucose we are competing with the bound glucose for the desired protein, releasing the protein from the column beads?
Thank you
TLDR: elution step, competition for ligands on beads using the same ligand?
I'm having trouble understanding how elution works in an ion exchange, specifically in a household water softener.
My understanding is the resin beads will have a negative charge (COO-). The resin beads are initially positively charged with sodium (Na+). When hard water flows through, the calcium (Ca2+) and magnesium (Mg2+) in the water has a stronger positive charge than the Na+; therefore, it will displace the Na+. Effectively pulling out the Ca2+ and Mg2+ out of the water.
Then, when you do the recharge of the beads/elution, you flow through a much higher concentration of Na+ and those will reverse the process and displace the Ca2+ and Mg2+.
But I don't understand how the higher concentration Na+ is able to displace the Ca2+ and Mg2+. I thought the Ca2+ and Mg2+ is bound stronger than Na+. The analogy I have in my head is a monkey attached to a tree branch. If an ape comes along, it's stronger than the monkey, the monkey's got to go. It shouldn't matter that bunch of monkeys come along, there's only room for 1 primate and a higher concentration of monkeys isn't going to help. None of them are individually stronger than the ape.
Hi friends,
Just wanted to confirm something... is that spike in fluorescence at the tail end due to all the fluorescent dye molecules that weren't incorporated into the liposomes? Because I assume they're the smallest component of the mixture, so they would come out last.
Thanks!
Edit: Original Question
https://preview.redd.it/zoz0ubfekfg41.png?width=640&format=png&auto=webp&s=9ecd1e9ca379d6a27aa8d635a028a77e49716181
https://preview.redd.it/8pf929rt4eg41.png?width=506&format=png&auto=webp&s=ed264e0b389f94550285cf493d6a933898c039ec
Hi Scientists,
I am looking to purify HIV neutralizing antibodies from human plasma using a protein A spin column kit (cosmo bio). The kit can only purify up to 0.4 mg of IgG. Other relevant information such as the binding and elution pH is not provided by the manufacturer.
To avoid overloading my column, I use 100 uL of plasma for IgG purification. However, the neutralizing ability of the subsequent IgG is very low relative to that of unfractionated plasma. I have checked the plasma flowthrough for any neutralization activity but there's none. I have also tried dialysis against PBS to no avail. So far I have been stuck at this stage for almost 4 weeks now.
I was wondering if the problem could be the binding capacity of the kit. Do you think a binding capacity of 0.4 mg is too low to elute a meaningful titer of my antibody of interest?
Have you experienced a similar problem with the kit (or another manufacturer's kit)? How did you go about?
Hello,
Can someone please explain to me how to figure out an elution order in reverse phase chromatography? And how pH comes into it please? I'm not getting too much useable information on Google? Many thanks :)
Hey everyone. I have a quick question about GST purification.
I am currently in the process of self-teaching how to do a GST purification. My end goal is take my GST purified protein and mix it with my His purified protein to test for an interaction.
I have successfully cloned my gene in the pGEX vector and purified it using glutathione beads and gravity filtration. My coomassie gel looks really good with multiple elution lanes expressing my protein with little background.
I am looking for advice now on what to do next. I can’t cleave off the GST tag because my protein domain of interest is only 8 kDA and I will need to probe using an anti-GST antibody.
I know that I most likely will need to remove the Reduced glutathione and move to a new buffer. My question is how? Does anyone that have more experience with this has a recommendation on protocol and reagents to do this?
Thanks in advance and let me know if I can add any more info!
Hi comrades, I am trying to do immunoprecipitation using streptavidin Dynabeads. My ab is biotinylated. So when I elute the antigen from the beads-streptavidin-biotinylated ab-antigen complex using the conventional glycine elutiom buffer, will the ab be eluted as well? I know the avidin-biotin is strong but just wanna make sure...
I don't wanna use the crosslinker or surface activated beads since they are expensive and we don't have them...
Hey guys, any have experience with a basic NiNTA elution buffer turning pink? Made some fresh buffers last week
NiNTA wash buffer: 50mM Tris-Cl, pH7.5 100mM NaCl 1mM DTT 10mM imidazole 5%glycerol
NiNTA elution buffer:: 50mM Tris-Cl, pH7.5 100mM NaCl 1mM DTT 300mM imidazole 5%glycerol
Anyway, my elution buffer (and only my elution buffer) got into the valentine’s day spirit and turned pink over the weekend. Any experience with this?
