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Natural Science Forum / Biology / Microbiology / December 2005How to rehydrate the IPG strip
Dear Sir or Madam:: I am Sheng Jiang. I now use Immobilized pH Gradient (IPG) to determine the isoelectric point (pI) of beta-lactamase. I used the mixture of 300µl urea ,CHAPS and 100µl the beta-lactamase to rehydrate the IPG strip. But I failed to stain the IPG strip using nitrocefin. Can you tell me how to rehydrate the IPG strip when determine the pI of beta-lactamse? I am looking forward eagerly for you reply. Sincerely yours, Sheng Jiang 2005-12-13 > Dear Sir or Madam:: > I am Sheng Jiang. I now use Immobilized pH Gradient (IPG) to determine [quoted text clipped - 9 lines] > Sheng Jiang > 2005-12-13 Hi Sheng Jiang When we want to do Iso-electric focusing using IPG strips for beta-lactamases, we simply rehydrate the strip using beta-lactamase (10 ul) & distilled H2O 290 ul), and this works fine. These volumes are right for the particular strips we use, I expect you can alter it accordingly, & increase/decrease total volume and the amount of beta-lactamase added etc if more enzyme is required e.g. if it doesn't hydrolyse nitrocefin very well. We never add urea - it has a tendency to denature enzymes!. I take it your enzyme does hydrolyse nitrocefin? (I do know people who tried this method with b-lactamases that wouldn't hydrolyse nitro!) If your enzyme is not great at hydrolysing nitrocefin there are other methods for staining IEF strips to visualise b-lactamases, I can't remember exact details, but they are described in the papers on protein characterisation of ImiS and CphA Aeromonas metallo-b-lactamase enzymes. Tam Dear Tam: Thank you very much! I use two EP tubes for a test: The first one: 3µl ß-lactamase solution + 3µl nitrocefin 0.5mg·ml-1. It becomes purple. The second one: 6µl ß-lactamase solution + 9µl urea and CHAPS solution + 6µl nitrocefin 0.5mg·ml-1 . It don not become purple. It do not change the color. You are right! I should not add urea or CHAPS. Only water can do! Do you think so? I have other questions: 1.Do you know what the electric voltage and the hours are needed? 2.Nitrocefin cannot be made in China. I only can buy it from Oxoid company, UK. The price is very high! I only buy 1mg. I know the concentration 0.5mg·ml-1 can stain the gel. But I only have 1mg. Do you know whether the lower concentration can do? I am looking forward for your reply. Sincerely yours, Sheng Jiang 2005-12-15 > Dear Sir or Madam:: > I am Sheng Jiang. I now use Immobilized pH Gradient (IPG) to determine [quoted text clipped - 9 lines] > Sheng Jiang > 2005-12-13 don't you think that if you are doing ief, you need a pH gradient? doesn't it make sense then that you would not want to use things like urea and CHAPS, as urea affects pH and CHAPS is a buffer? maybe I am on glue, but it sounds to me like your reconstitution is going to ruin your expt. |
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