Concentrating conditioned medium from cells - (Sep/22/2005 )
Hello everybody!
I would like to concentrate conditioned medium from cells and use it for western blot. I used speedvac to concentrate the medium, but i had troubles running the concentrate on a PA gel. I got a suggestion to dialyse the medium first. Does anybody have any experience in this technique?
Thanks,
Snunit
i did once lyophilization however it wasnt for western. i think you can try it because you dont need to keep biological activity of the proteins.
A potential problem with dialysis is protein precipitation in the dialysis unit because of (possible) insolubility of your protein in low salt concentration.
Your best guess could be ultrafiltration micro-columns. These concentrate your sample by centrifugation, while keeping salt concentration constant. Just make sure the column are labeled "low protein binding", and that the membrane cut-off is well below the size of your protein. Oh, and watch for the tempature.
Could you be more specific about the "problems" you encounter on PA gel? This might help the readers' diagnosis.
Good luck.
Thank you very much for your suggestion. I'm trying out a millipore ultrafiltration columns centrifuging for 1 hour at 14.000 rpm at 4 C.
The problems were following: sample concentration was 0.6 microgram/microliter. I loaded 15 microgram (25 microliters+25 microliters of loading buffer. it's actually the maximum one can load in this type of machine). The samples didn't enter the concentrating gel, it looked like the slots were overloaded.
I would like to concentrate conditioned medium from cells and use it for western blot. I used speedvac to concentrate the medium, but i had troubles running the concentrate on a PA gel. I got a suggestion to dialyse the medium first. Does anybody have any experience in this technique?
Thanks,
Snunit
I'm looking into precipitating the proteins from media in 5 volumes of 100% Ethanol at -20 degrees C for 24hrs, spin full speed 10mins at 4 degrees C. Then wash the pellet in 5 volumes 90% ice cold ethanol, spin full speed for 5 mins at 4 degrees C. Then aspirate, air dry and resuspend pellet in whichever buffer is best suited to you (Urea, SDS, acetic acid) . I know it works for precipitaing proteins out of 4M GITC, don't see why it wouldn't work for growth media. Any comments or past experience with this methodology would be greatly appreciated.
Lyophilization is also good if you have the kit to do it, however I find it easier to resolubalise all the protein if it is in a pellet rather than all over the tube
Have a look at the methods on this web site http://www.ls.huji.ac.il/~purification/Pro...on.html#Ethanol
Hello Mikemessy,
Thank you very much for your suggestions!
Actually in a mean time I managed to solve the problem . I used Microcon Centrifugal Filter Device of Millipore (Microcon YM-10, Cat No: 42407), which gave fantastic results. Only one thing one has to be aware of: do not use media with more than 1 % FCS in it. For the rest this method is easy, relatively cheap, takes only 1 hour of centrifugation.
If you have any questions, do not hesitate, contact me.
Snunit
I'm looking into precipitating the proteins from media in 5 volumes of 100% Ethanol at -20 degrees C for 24hrs, spin full speed 10mins at 4 degrees C. Then wash the pellet in 5 volumes 90% ice cold ethanol, spin full speed for 5 mins at 4 degrees C. Then aspirate, air dry and resuspend pellet in whichever buffer is best suited to you (Urea, SDS, acetic acid) . I know it works for precipitaing proteins out of 4M GITC, don't see why it wouldn't work for growth media. Any comments or past experience with this methodology would be greatly appreciated.
Lyophilization is also good if you have the kit to do it, however I find it easier to resolubalise all the protein if it is in a pellet rather than all over the tube
Have a look at the methods on this web site http://www.ls.huji.ac.il/~purification/Pro...on.html#Ethanol
[/quote]
If you have any questions, do not hesitate, contact me.
Snunit
I'm glad it worked...