现在需要把yeast extract里面的protein含量测出来。我们要的是其中的peptide。但 是想知道里面protein的杂质的量。请问什么assay可以测出来?Bradford protein assay和Bio-rad protein assay 应该是区分不出peptide和protein的吧?谢谢指教!
Thanks for your suggestion! But we currently already use HPLC to quantify our peptide product. Protein cannot be quantified by FPLC\HPLC, right? I'm not sure about Mass spec though.
【在 s******y 的大作中提到】 : Using FPLC, HPLC, or Mass Spec 来分离大分子和小分子。 : 又快又简单,就是对器材要求高一些。
【在 e****e 的大作中提到】 : Thanks for your suggestion! But we currently already use HPLC to quantify : our peptide product. Protein cannot be quantified by FPLC\HPLC, right? I'm : not sure about Mass spec though.
According to your opinion, I should use Bradford assay to determine total protein and peptide. I will get the total amount of my peptide product from HPLC. Then the protein amount is total protein and peptide minus the amount of my peptide product. Dialysis is not necessary, right?
Gel Filtration Chromatography (Size-exclusion chromatography)
【在 e****e 的大作中提到】 : Thanks for your suggestion! But we currently already use HPLC to quantify : our peptide product. Protein cannot be quantified by FPLC\HPLC, right? I'm : not sure about Mass spec though.
p*n
44 楼
他是对的,如果几百回复当时 能解决问题的话,我不会到8年后 才说出这些话。
【在 p******n 的大作中提到】 : 他是可怜我,他看到我确实有麻烦。
e*e
45 楼
No. I came from biochemistry labs. So can I get an conclusion that there is no a method can detect only the amount of protein but peptide? Of course I have a method to detect the amount of the peptide product.
【在 e****e 的大作中提到】 : No. I came from biochemistry labs. So can I get an conclusion that there is : no a method can detect only the amount of protein but peptide? Of course I : have a method to detect the amount of the peptide product.
r*n
50 楼
是的。我知道他会。我现在应该基本具备尝试CHANDING了,只是我还是有点娇气。
【在 p******n 的大作中提到】 : 他答应过你的事,他会做到,我信,你要CHANDING。
e*s
51 楼
上样量太大了!?有条件弄长点的柱子。
【在 e****e 的大作中提到】 : Gel Filtration Chromatography shows a flat peak starting from the void : volumn to the end. Yeast extract is quite dirty.
I already got what I need to know. My question is very simple. That is, is there any method can measure only protein but no peptide without further seperation or purification? I thought the answer should be "No" before I came here. But I am not so sure since I never think about it before. When I worked in purification labs, we always used OD280 to determine protein concentration since the protein is quite pure. When I worked in molecular biology lab, we always used Bradford to determine the protein concentration of whole cell lysate. When I worked in manufacturing labs, HPLC is used to determine small molecular concentration. But I never think all these detect methods together. All the above suggestions confirm my answer since the definition between protein and peptide is not distinct. Of course I know the purification method for seperating the peptide from proteins or the yeast extract well. So I can't help smiling when I saw those that suggest to do dialysis.
【在 e****e 的大作中提到】 : I already got what I need to know. My question is very simple. That is, is : there any method can measure only protein but no peptide without further : seperation or purification? I thought the answer should be "No" before I : came here. But I am not so sure since I never think about it before. When I : worked in purification labs, we always used OD280 to determine protein : concentration since the protein is quite pure. When I worked in molecular : biology lab, we always used Bradford to determine the protein concentration : of whole cell lysate. When I worked in manufacturing labs, HPLC is used to : determine small molecular concentration. But I never think all these detect : methods together.
我明白你的意思了。 你本来就是想看看有什么最新的特别fancy 的技术可以不通过分离而直接 挑出peptide or protein来特异反应啊。 那还真的没有这种技术。 因为蛋白本来就和peptide 没有啥严格区别。
is
【在 e****e 的大作中提到】 : No. I came from biochemistry labs. So can I get an conclusion that there is : no a method can detect only the amount of protein but peptide? Of course I : have a method to detect the amount of the peptide product.
As I said before, I've worked on protein projects for a long time. But I never think too much. Once a person who never works on this field ask such an question, I lost for a moment. Just like when a kid saw a piece of white paper turns to black when the paper was burned, he asked why the white turns to the black. I told the kid the fire make the paper to carbon. Then the kid said: "So the paper is not carbon?" I suddenly realized the paper is also made of wood and it should be carbon. Then I need to think more and explain more. hehe, I am not sure if this is other similar example. If not, then just for sharing some funny things. Seriously, I don't understand why you think 3kD peptides will be difficult to be over-expressed. I didn't work on this peptide's expression. But according to my previous experience, couples of 5 to 9 kD proteins in my hand are very easy to over-express in E.Coli.
【在 e****e 的大作中提到】 : As I said before, I've worked on protein projects for a long time. But I : never think too much. Once a person who never works on this field ask such : an question, I lost for a moment. Just like when a kid saw a piece of white : paper turns to black when the paper was burned, he asked why the white turns : to the black. I told the kid the fire make the paper to carbon. Then the : kid said: "So the paper is not carbon?" I suddenly realized the paper is : also made of wood and it should be carbon. Then I need to think more and : explain more. hehe, I am not sure if this is other similar example. If not, : then just for sharing some funny things. : Seriously, I don't understand why you think 3kD peptides will be difficult
This depends on what peptides you are talking about. Over the years, I have produced multiple peptides (some are qualified as proteins, e.g. ww domains) between 3-5 KDa in E. coli: some of them are disordered, some are single helix and some are a few beta-strand. Many of them have very high yields.
hi, buddy, what define a protein or peptide, is it the size? 50-100aa?
have domains)
【在 b******y 的大作中提到】 : This depends on what peptides you are talking about. Over the years, I have : produced multiple peptides (some are qualified as proteins, e.g. ww domains) : between 3-5 KDa in E. coli: some of them are disordered, some are single : helix and some are a few beta-strand. Many of them have very high yields.
If you have a 50 a.a peptide with no particular 2nd structure, I call it a peptide. Some polypeptides, like ww domains, have only about 40 a.a. (3 short beta strands packed in an antiparallel fashion), which can bind proline-rich peptide ligands. I can call them proteins or at least protein domains. I believe there are a few domains that are of comparable size or even smaller than ww domains. Don't remember what they are called on top of my head. As we all know, it is so trivial to translate DNA to a.a. sequence. However, it is harder to define ORF just based on translation into peptide because a real ORF has to code a number of elements: promoter sequence for transcription, initiation ATG-codon, ribosome entry site, poly-A signal, etc .
but I was thinking about determining the existence of peptide and then reverse to find potential ORF...feasible or not, any potential/progress in this area
of However, a
【在 b******y 的大作中提到】 : If you have a 50 a.a peptide with no particular 2nd structure, I call it a : peptide. Some polypeptides, like ww domains, have only about 40 a.a. (3 : short beta strands packed in an antiparallel fashion), which can bind : proline-rich peptide ligands. I can call them proteins or at least protein : domains. I believe there are a few domains that are of comparable size or : even smaller than ww domains. Don't remember what they are called on top of : my head. : As we all know, it is so trivial to translate DNA to a.a. sequence. However, : it is harder to define ORF just based on translation into peptide because a : real ORF has to code a number of elements: promoter sequence for
Finally to LZ, you can quantify your product using HPLC at any stage of your purification. Meanwhile measuring total protein including protein, peptide and your product using branford or methods alike, you will know the enrichment or purity at any stage. I believe this is more or less how your company is doing it. What makes me agitated is: Even if you can magically distinguish 3 KDa peptides from 1-2.9 KDa and 3.1 KDa-infinite, you still don't know whether they are purely from your product or partial from impurity. You knew this all along!? Why still keep bothering us?
You mean MS whole cell proteome and then determine ORF accordingly. Good thought. I have to wait for bioinformatics experts to answer this question.
【在 n********k 的大作中提到】 : but I was thinking about determining the existence of peptide and then : reverse to find potential ORF...feasible or not, any potential/progress in : this area : : of : However, : a
【在 b******y 的大作中提到】 : Finally to LZ, you can quantify your product using HPLC at any stage of your : purification. Meanwhile measuring total protein including protein, peptide : and your product using branford or methods alike, you will know the : enrichment or purity at any stage. I believe this is more or less how your : company is doing it. : What makes me agitated is: : Even if you can magically distinguish 3 KDa peptides from 1-2.9 KDa and 3.1 : KDa-infinite, you still don't know whether they are purely from your product : or partial from impurity. You knew this all along!? Why still keep : bothering us?
Thanks for sharing the info. Clearly, academia is totally different from industry. Out of curiosity, what kind of and how many drugs are made of peptide?
【在 b******y 的大作中提到】 : Thanks for sharing the info. Clearly, academia is totally different from : industry. : Out of curiosity, what kind of and how many drugs are made of peptide?
利用人工智能免疫系 likes CD44 plus Hyaluronan (hyaluronic acid, HA) is a linear naturally occurring polysaccharide 钓3kDa左右的多肽的鱼 可以不通过分离而直接挑出peptide or protein来特异反应--just matrix two cents. References: 1) Erickson M, Stern R. Chain gangs: new aspects of hyaluronan metabolism. Biochem Res Int.2012:893947. //www.ncbi.nlm.nih.gov/pubmed/22216413 and 2) Chaim OM et al.(2011) Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes. Toxins (Basel). 3: 309-344 Abstract Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5-40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein , we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins. //www.ncbi.nlm.nih.gov/pubmed/22069711 3) Vaaje-Kolstad G et al. (2010) An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science. 330: 219-222. //www.ncbi.nlm.nih.gov/pubmed/20929773 more please refer one mentor Robert Stern of UCSF for Hyaluronan (hyaluronic acid, HA) and CD44 his publications summary: //www.researchgate.net/researcher/39036610_Robert_Stern or other labs relative to this topic: //www.ncbi.nlm.nih.gov/pubmed?db=pubmed&cmd=link&linkname=pubmed_pubmed_ citedin&uid=12385018 ----- [ 33 ] 发信人: sunnyday (飞鱼), 信区: Biology 标 题: Re: 什么protein assay可以区分protein和peptide? 发信站: BBS 未名空间站 (Wed Jun 20 10:48:23 2012, 美东) 我明白你的意思了。 你本来就是想看看有什么最新的特别fancy 的技术可以不通过分离而直接 挑出peptide or protein来特异反应啊。 那还真的没有这种技术。 因为蛋白本来就和peptide 没有啥严格区别。
is
【在 e****e 的大作中提到】 : No. I came from biochemistry labs. So can I get an conclusion that there is : no a method can detect only the amount of protein but peptide? Of course I : have a method to detect the amount of the peptide product.
【在 l**********1 的大作中提到】 : 利用人工智能免疫系 likes CD44 plus Hyaluronan (hyaluronic acid, HA) is a : linear naturally occurring polysaccharide 钓3kDa左右的多肽的鱼 : 可以不通过分离而直接挑出peptide or protein来特异反应--just matrix two cents. : References: : 1) : Erickson M, Stern R. : Chain gangs: new aspects of hyaluronan metabolism. : Biochem Res Int.2012:893947. : //www.ncbi.nlm.nih.gov/pubmed/22216413 : and