2010-09-09T14:41:28-07:00 ATOM Feed of SilkWise.com (Forum: Antagene, Inc) Copyright by this site owner SilkWise.com Editor http://www.silkwise.com/content/rsssummary mvnForum Feed Generator 1.2 GA http://www.silkwise.com/content/viewthread_thread,14790 2009-11-05T04:10:31-08:00 2009-11-05T04:10:49-08:00 How it works? mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14789 2009-11-05T04:09:26-08:00 2009-11-05T04:09:26-08:00 We did provide you with antibody ELISA test against phospho-peptide and non-phospho peptide, it will be upon to you to test with corresponding phospho- proteins and non-phospho proteins by your application for example by Western blot. But bear in mind, it's very unpredictable if a designed peptide antibody is against the native protein. There is big gap between peptide and whole protein due ... mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14788 2009-11-05T04:08:32-08:00 2009-11-05T04:08:32-08:00 Below is our ELISA protocol. Please bear mind, using the correct second antibodies to do your ELISA tests. Elisa Protocol Materials: 1.Solid phase immobilized Ag: Microtiter wells coated with 1ug/100ul/well of peptide BSA are used to capture antibody. 2.Antiserum 3.HRP-conjugated goat anti-rabbit IgG (Pierce). 4.Diluent for antibody or enzyme conjugate: 2% BSA in PBS. ... mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14787 2009-11-05T04:06:29-08:00 2009-11-05T04:06:46-08:00 How long the titer will remain after last booster? How to reduce the background and to have a better chance to see reaction with native protein? mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14786 2009-11-05T04:02:59-08:00 2009-11-05T04:02:59-08:00 Yes, we have the affinity-purification service available. The price is $600 for purification of 100ml antiserum and $500 for 50ml, which includes 5ml of affinity-gel conjugated 10mg of the peptide, purified IgG and ELISA data on every stage of the purification, and the used antiserum will be return to you together. The yield will be 5-10mg purified antibody from 50ml of antiserum in most cases, ... mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14785 2009-11-05T03:55:03-08:00 2009-11-05T03:55:26-08:00 How to get a single band in Western Blot test with your antibody? mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14784 2009-11-05T03:53:47-08:00 2009-11-05T03:53:47-08:00 To make a good antibody, some researchers try to generate the antibodies by different sites on the protein sequence, and ideally select 3 sites, N-terminal, C-terminal and the middle, to do the immunization. Believe or not, the different site peptide will give you different results. So, it's not surprise for that author used different antibodies to do the different experiments. Another option ... mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14783 2009-11-05T03:52:04-08:00 2009-11-05T03:52:24-08:00 In respect to use the conjugates to immunize mice for the following hybridoma production, most people used to use 50 ug of a purified antigen per injection per mouse. Because the antigen concentration in these preparations is unclear, how much the KLH from the conjugate's tubes should be used per injection per one mouse? How many injections do you expect to induce specific response? Do you ... mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14782 2009-11-05T03:47:16-08:00 2009-11-05T03:47:16-08:00 Here is the protocol of our KLH-peptide conjugation. Briefly, we weight 15mg KLH reacting with SMCC linker in PBS. After PD-10 column removed unbound SMCC, 15mg of free peptide were added to the SMCC linked KLH for coupling overnight. Finally, the free peptides were removed by exhaustedly dialysis. mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14781 2009-11-05T03:46:13-08:00 2009-11-05T03:46:13-08:00 The yield of affinity-purification is very diversity in different rabbit and is depending on the immune response of the individual animal to the antigen. We got 5 to 10 mg of 96-99% purified IgG from 50ml antiserum in most case. The ELISA range of the purified antibodies is 1.5-3.0 with 1:1k dilution from 1mg/ml, over 80% of antibodies is above 2.0 ELISA titers. The efficiency is almost 100% for ... mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14780 2009-11-05T03:45:33-08:00 2009-11-05T03:45:33-08:00 We used to cut a piece of the yolk and weight it, then add the dd water into the tube, break the yolk by a glass bar or the pipette and vortex. Also, you may bring the yolk back by put the frozen tube into the water bath at 37-50oC, transfer the viscous yolk by a blade. The yolk is always viscous, you should weight it, and dilute it by the w/v mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14779 2009-11-05T03:44:29-08:00 2009-11-05T03:44:29-08:00 The anti-chicken second antibodies are available in most company. It's from MolecularProbe company which I used for the Cell staining and Western blot. The simplest treatment is adding 10 folds of dd water at pH5.2 to the egg yolk, incubating for 30-60 minutes at RT, and then centrifuge for 10-20 min at top speed of your eppendorf. The supernant can be used to do western and ELISA after ... mikemao@autogeneinc.com http://www.silkwise.com/content/viewthread_thread,14778 2009-11-05T03:31:35-08:00 2009-11-05T03:37:17-08:00 some of them make good mix, other just stuck to the wall. mikemao@autogeneinc.com