Re: [ccp4bb] Lost my protein

Is your protein sticking to the membrane or crashing/aggregating from the concentrating? Try to find a buffer condition in which your protein is more soluble and less aggregation prone. You can try varying salt concentration, pH, and inclusion of additives like glycerol or detergent. Ho UC Berke

Re: [ccp4bb] Lost my protein

bulletin board on behalf of Sophia Tsai > Sent: Wed 5/6/2009 7:12 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Lost my protein > > Hi everyone, > It seems that our lab has experienced issues with precipitation of some > proteins (not all) with the Amicon concentrator

Re: [ccp4bb] Lost my protein

@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Lost my protein Hi everyone, It seems that our lab has experienced issues with precipitation of some proteins (not all) with the Amicon concentrators. For those, we have started using VivaSpin concentrators. That seems to work fairly well with those that have issues with

Re: [ccp4bb] Lost my protein

Dear Zhang, Perhaps you can try to concentrate your protein using PEG2. Put your protein into dialysis tubing and bury it with PEG2. You should check it every 30-60 min. Sincerely, Li Hi all, during concentrating my protein, using Amicon Ultra centrifugal filter

Re: [ccp4bb] Lost my protein

Hi everyone, It seems that our lab has experienced issues with precipitation of some proteins (not all) with the Amicon concentrators. For those, we have started using VivaSpin concentrators. That seems to work fairly well with those that have issues with the filters. You may want to give it a try.

Re: [ccp4bb] Lost my protein

PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Lost my protein Hi. I have experienced similar problems with the Amicon filters previously. I found that using a swing bucket rather than a fixed angle centrifuge and keeping the speed at 4000 g, the recommended speed, resulted in better yields

Re: [ccp4bb] Lost my protein

Hi. I have experienced similar problems with the Amicon filters previously. I found that using a swing bucket rather than a fixed angle centrifuge and keeping the speed at 4000 g, the recommended speed, resulted in better yields. If you have a large volume to concentrate (>12 mL), using an Amico

Re: [ccp4bb] Lost my protein

Hi Yanming, I usually keep the follow through before I know nothing is in it. Also you may want to suspend your protein solution every 5-10 min to avoid precipitation because the concentration gradient forms rapidly during centrifugation. If all of these do not help, just try concentrators wit

Re: [ccp4bb] Lost my protein

Hi Yanming, you can try adding 1 M of urea, 1% detergent (OG,DM) that often helps to keep proteins in solution. Best wishes Kornelius On Tue, 5 May 2009 09:06:09 -0700 yanming Zhang wrote: > Hi all, > > during concentrating my protein, using Amicon Ultra centrifugal filter > devices, 5000g

Re: [ccp4bb] Lost my protein

Check the speed--for the 15mL devices, ~3000 x g is the cutoff, I think. You may be breaking the membranes, and your protein is simply in the bottom of the concentrator. JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Progr

Re: [ccp4bb] Lost my protein

Hi, assuming that 1) you used the right MW cut-off and 2) the membrane is intact, you probably "pelleted" your protein onto the membrane. This often happens if the protein has a limited solubility in the buffer you are using. Remember, while concentrating your protein, you are actually "trave