rimwehbeZ0 Posted February 28, 2019 Report Share Posted February 28, 2019 Please, does anyone know a protocol to purifiy an enzyme from crude honey bee venom? There are 2 major components of honey bee venom which are Melittin and PLA2; both seems always attached to one another... how can i purify PLA2?? Quote Link to comment Share on other sites More sharing options...
Vmedvil2 Posted March 1, 2019 Report Share Posted March 1, 2019 (edited) Please, does anyone know a protocol to purifiy an enzyme from crude honey bee venom? There are 2 major components of honey bee venom which are Melittin and PLA2; both seems always attached to one another... how can i purify PLA2??Here is a article on enzyme purification, with an extended PDF.https://www.biotecharticles.com/Applications-Article/Methods-of-Purification-of-Enzymes-583.html Edited March 1, 2019 by VictorMedvil Quote Link to comment Share on other sites More sharing options...
rimwehbeZ0 Posted March 3, 2019 Author Report Share Posted March 3, 2019 Can anyone please give me an efficient protocol for cation exchange chromatogprahy in order tu purify PLA2 from honey bee venom using a LC-SCX column?? please and thank you. Quote Link to comment Share on other sites More sharing options...
Vmedvil2 Posted March 3, 2019 Report Share Posted March 3, 2019 (edited) Can anyone please give me an efficient protocol for cation exchange chromatogprahy in order tu purify PLA2 from honey bee venom using a LC-SCX column?? please and thank you. Here is the teacher's guide book on Cation Exchange Chromatography for peptides which are protein fragments. https://www.gbiosciences.com/image/pdfs/protocol/BE-415_protocol.pdf Here are two Powerpoint on the process of doing it. https://slideplayer.com/slide/10135169/ https://www.slideserve.com/kemp/ion-exchange-chromatography Here is a journal on the process of doing chicken egg. https://www.sciencedirect.com/science/article/pii/S0021967305013506 Edited March 3, 2019 by VictorMedvil Quote Link to comment Share on other sites More sharing options...
rimwehbeZ0 Posted March 4, 2019 Author Report Share Posted March 4, 2019 thanks a lot! Quote Link to comment Share on other sites More sharing options...
fahrquad Posted March 5, 2019 Report Share Posted March 5, 2019 (edited) I have considered creating an alternate identity so I can post arcane questions with one identify and provide textbook answers with the other to appear particularly bright. Then I could use the one identity to give positive rankings to the other...or I could just get a f***ing life. Edited March 5, 2019 by fahrquad Quote Link to comment Share on other sites More sharing options...
rimwehbeZ0 Posted March 5, 2019 Author Report Share Posted March 5, 2019 if i have a peak of 140 mAU protein instensity on a RP-HPLC Chromatogram which I do not know its approximative weight... is there a way to know its approximative weight based on the peak? cause the MS machine is not working Quote Link to comment Share on other sites More sharing options...
Vmedvil2 Posted March 6, 2019 Report Share Posted March 6, 2019 (edited) if i have a peak of 140 mAU protein instensity on a RP-HPLC Chromatogram which I do not know its approximative weight... is there a way to know its approximative weight based on the peak? cause the MS machine is not working You would have to know how long the machine was running in order to determine total weight based on intensity of the proteins. What is your injected mass and the length of the protein that is venom and the length of the protein that is the other protein in base pairs. (Injected mass) (Length of Bee venom Protein in Base pairs)/((Length of Bee venom Protein in Base pairs) + (Length of Other Protein in Base Pairs)) = (Mass of Bee Venom) So let's say you injected a mass of 30 grams into the devices and the length of the bee venom protein in base pairs is 1500 bp and the length of the other protein is 800 bp, then your mass of bee venom from the reaction would be (30 * 1500)/(1500 + 800) = (Mass of Bee venom in Grams) approximately. This is a simple ratio that determines that amount of mass is bee venom versus other proteins. If that method is not working for you in protein purification you can always take the genetic route to protein purification and use a Viral Vector to directly modify the bees that you are working with to have the venom genetically engineered to be separated from the other protein using a Bee-Borne Viral Vector with CRISPR in it to genetically cleave on both sides of the protein making it into two parts for nano-scale purification of the protein such as LTR5' - Cas9 - LTR3' , Pol - Gag - ENV HIV-1 - Glycoprotein of Bee Infecting Virus such as Cripaviridae type viruses. Or if you want to go for the synthetic synthesis of your protein you could always isolate the gene that synthesizes the Bee Venom and insert the gene into a plasmid then fuse it with a Machine Bacteria like Metamorphic Gel with Ribosomes to have a organism produce the Bee Venom automatically seperated from the other proteins and isolate the gene instead of the protein into a Machine Bacteria that only produces purified Bee Venom from the PLA2 gene. In this form your Plasmid itself would be worth 250,000$ to 1 million dollars by itself because of purification of the gene, I have 10 which produce Nano-fabricated Viral Vectors in this form. MV-3 Tri-valent Phage and Machine Bacteria Producer Plasmid Edited March 6, 2019 by VictorMedvil Quote Link to comment Share on other sites More sharing options...
rimwehbeZ0 Posted March 17, 2019 Author Report Share Posted March 17, 2019 Thank you so much, but our lab does not have the requirements to do such things, I really do need to have an efficent protocol for the separation of my bee venom using chromatography on SCX (strong cation exchange) column! like what are the buffers that i need to use etc...?? Quote Link to comment Share on other sites More sharing options...
A-wal Posted March 17, 2019 Report Share Posted March 17, 2019 I have considered creating an alternate identity so I can post arcane questions with one identify and provide textbook answers with the other to appear particularly bright. Then I could use the one identity to give positive rankings to the other...or I could just get a f***ing life. Interesting. Bwal 1 Quote Link to comment Share on other sites More sharing options...
Vmedvil2 Posted March 18, 2019 Report Share Posted March 18, 2019 Thank you so much, but our lab does not have the requirements to do such things, I really do need to have an efficent protocol for the separation of my bee venom using chromatography on SCX (strong cation exchange) column! like what are the buffers that i need to use etc...?? Honestly, you will have to look around I dunno alot about Chromatography other than what was posted, I don't use that process very often it was sort replaced by gene isolation as described in my opinion, You don't need to isolate the protein anymore just the gene that makes the protein for such things which is apart of modern Biotechnology that has simplified that process. Personally, I don't use Chromatography very often. Quote Link to comment Share on other sites More sharing options...
rimwehbeZ0 Posted March 19, 2019 Author Report Share Posted March 19, 2019 Honestly, you will have to look around I dunno alot about Chromatography other than what was posted, I don't use that process very often it was sort replaced by gene isolation as described in my opinion, You don't need to isolate the protein anymore just the gene that makes the protein for such things which is apart of modern Biotechnology that has simplified that process. Personally, I don't use Chromatography very often. I get it thank you once more. My aim is to purify PLA2 from crude bee venom but there are a lot of obstacles facing me due to the inadequate facilities in our lab sadly, the problem with purifying the PLA2 is that it is always somehow attached with melittin, if you know someone who can help me on some sort of experiments ( that might be applicable in our lab) it would be really great... thank you once more. Quote Link to comment Share on other sites More sharing options...
fahrquad Posted March 20, 2019 Report Share Posted March 20, 2019 (edited) Interesting. Cute. Am I the only singularity here? Edited March 20, 2019 by fahrquad Quote Link to comment Share on other sites More sharing options...
A-wal Posted March 20, 2019 Report Share Posted March 20, 2019 Cute. Am I the only singularity here? Tis not my fault. I can't upload attachments with my main profile, think I reached my limit. Small limit. Quote Link to comment Share on other sites More sharing options...
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