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| I looked for several books and protocols, however I couldn't figure out. I have high background in IP and there is not even candidate band either. I didn't know the result is right because I don't know how to do IP very well. In the protocols, there are just simple words.. "Add lysis buffer 0.5~1ml and centrifuge 2500g and discard sup. Do same washing more than 4 times. These are what I would like to know 1. Does it matter to use lysis buffer or lysis buffer with inhibitor during washinhg step? 2. do I have to vortex or shake up and down everytime after adding lysis buffer? 3. If I added 1ml buffer, do I have to take out almost 1ml or a lot less after centrifugation? 4. What is efficient way to take out buffer with not touching bead? Please, help me ... I am stucking in this IP problem for several weeks. Thank you, |
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The foremost problem is the SDS it will dissociate the antigen-antibody complex, next the salt concentration from Tris, NaCl and NaDeoxycholate can also dissociate the capture complex. Try PBS-Tween for a wash. |
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| Since I use specific beads, I couldn't use IP kit, I placed an order for spin cup not but IP kit. For the spin cup, putting the resin-sample, cenrifuge and elute sample. Am I right? Also, I am going to use PBS-tween for washing buffer rather than RIPA. Thank you for all your help. Have a nice Holiday. |
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| You can try washing with PBS + 2% Triton X-100. Add wash buffer, vortex then centrifuge at max speed in a table-top sized centrifuge for ~1min at 4 degrees- repeat another 2x. After last wash remove as much buffer as you can without disturbing beads. Another way to remove buffer is to set up a vacuum (using standard vacuum on a lab bench) and attach a syringe and fine needle. Just make sure the vacuum goes through a beaker of some description to dispose of the liquid if doing this way. |
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