 |
 | Home | Contact Us | FAQ | Search & Site Map | Link to Us |
 | Sign In | Join | Other 45 Sites in Network |
Natural Science Forum / Biology / Microbiology / November 2005A Chinese student ask for help: lactamase, Escherichia coli , IEF
Dear Sir or Madam: I am a student of Sichuan University in the city of Chengdu, China. My name is Sheng Jiang. I major in infectious disease. I am studying beta-lactamase in China. I want to get some advice from you. Now I am piloting inhibitor-resistant TEM in China. I have collected about 20 Escherichia coli, which is resistant to amoxicillin-clavulanic acid and sensitive to cefozolin and cefoxitin. Now I want to determine the isoelectric point (pI) of the ß-lactamase. But non of us have ever done it. So I want to get your advice about how to determine the isoelectric point of the ß-lactamase and how to prepare the ß-lactamase. 1. I want to prepare the ß-lactamase by means of ultrasonic treatment. Do you have the Standard Operation Procedure (SOP) about it? 2. I want to determine the isoelectric point of the ß-lactamase by means of Isoelectric Focusing (IEF). Do you have the Standard Operation Procedure (SOP) about it? 3. I want to use nitrocefin to stain gels for ß-lactamase. And I have bought 1mg nitrocefin from Oxoid Company, England. I have read some papers about Isoelectric Focusing. They said that the concentration of nitrocefin is 0.5mg/L. I think the concentration is very high. I want to know whether the lower concentration can work properly well or not. We have never prepare the ß-lactamase. Never we have determined the isoelectric point of the ß-lactamase. We want to get your advice. I am looking forward for you reply. My MSN: doctorjiangsheng@hotmail.com My E-mail:doctorjiangsheng@gmail.com Sincerely yours, Sheng Jiang 2005-10-10 > Dear Sir or Madam: > I am a student of Sichuan University in the city of Chengdu, China. My [quoted text clipped - 10 lines] > 1. I want to prepare the ß-lactamase by means of ultrasonic treatment. > Do you have the Standard Operation Procedure (SOP) about it? So you are lysing the cells by sonication?? sounds good. centrifuge the sonicated solution Determine which fraction the beta lactamase is in. Electrophorese the sup and pellet, look for your beta-lactamase protein band. To know which fraction it is in, you need to know the protein sequence of the beta lactamase in order to find a putative band of the same molecular weight. With this determined, you can use informatics to suggest what the pI is going to be, and based on that you will have a good idea if it works when you do the isoelectric focusing. The informatics tools can be found at expasy.org, there you can search for the protein sequence, and do pI calculations amongst many other tools related to proteins. > 2. I want to determine the isoelectric point of the ß-lactamase by > means of Isoelectric Focusing (IEF). Do you have the Standard Operation > Procedure (SOP) about it? Generally, you would be interested in something like an acrylamide gel which has pH gradients in it, ranging from acidic to basic. Most people do 2D gel electrophoresis to determine pI and molecular weight. the first step is to use the pH gradient gel, and proteins will migrate until they become neutral (at the pH where their pI is). Then use a standard gel attached to the gradient gel, and electrophorese in the perpindicular direction. By this method, you can determine the pI of proteins, by crossreferencing with molecular weight (you should know the molecular weight and theoretical pI from the informatics tools on expasy.org) Note that some proteins are modified after production, due to location addressing tags (ie. for excretion) getting chopped off. So the theoretical molecular weight is not always spot on. If you have more money, using capillary electrophoresis for iso-electric focusing apparently has far better reproducibility. I've heard you can rig up sequencing capillary electrophoresis units for this task, but haven't tried, as the CE I've used was dedicated to protein characterization. The CE IEF technique is better suited to purified proteins as well, so your sonicated mixture would probably not be suitable. > 3. I want to use nitrocefin to stain gels for ß-lactamase. And I have > bought 1mg nitrocefin from Oxoid Company, England. I have read some [quoted text clipped - 4 lines] > isoelectric point of the ß-lactamase. We want to get your advice. > I am looking forward for you reply. I know nothing about nitrocefin, but staining procedures generally come with the products, plenty of products reccommend you use more than you need, but if the protein you are after is only present in minute quantities, then using the recommended concentration may be critical to actually seeing results. If you know the beta lactamase is being over expressed relative to other proteins, then I'd say feel free to drop the concentration below the recommended value. Cheers, Mike Dear Mike McWilliams: I am Sheng Jiang and I have read your reply on goole sci.bio.mirobiology. I am very busy these days. And I am very late to reply you. I am very sorry for that. I am studying beta-lactamse now. And thank your advice. Sincerely yours, Sheng Jiang 2005-11-3 |
Reply to this Message |
 Sign In
 Join
 My Latest Posts My Monitored Threads My Blog My Photo Gallery My Profile My Homepage Start New Thread Enable EMail Alerts Rate this Thread
|
©2010 Advenet LLC Privacy Policy - Terms of Use
This website includes both content owned or controlled by Advenet as well as content owned or controlled by third parties.