Here is why we use the batch method. 1. We do it in the cold and do not have a working FPLC in a cold box or room. 2. Our volumes are large and do not have an easy way to load it using an FPLC. 3. The buffer volumes are greatly reduced using batch. This can be an issue with some detergents.
Concerning the concentration of imidazole in the wash, load, and elution buffers. 1. We determine that empirically using 0.1 - 0.2 ml columns - using a batch method. 2. The effective imidazole concentration is dependent on the resin, the pH, the protein, and the contaminants including lipids - (eg., phosphatidylcholine greatly inhibits binding to the column). 3. Once we have an answer, it seems to work every time - and we do it a lot. 4. Our primary failures have been do to improperly regenerated (or neglected) columns. On Thu, Aug 22, 2013 at 12:58 PM, Bosch, Juergen <jubo...@jhsph.edu> wrote: > Hi Ed, > > well if we hit the timer after lysis, say via cell disruptor then I have > my eluted protein in less than 1 hour, including 40 minutes batch binding. > If I'm pressed for time I bind only 20 minutes and then can have the > protein ready for the next step within 30 minutes after lysis. Some of you > are then still centrifuging and have not even started to load the column. > [Quick jump to last paragraph for the reason] > > I see the disadvantage more in occupying an Akta for something that could > be easily done without one. Also a pump with gradient mixer may be an > alternative. > But every protein is different and you should obey to your protein and not > stick to what you think is best, trial and error and hopefully moving fast > to some conclusions on how to best purify your protein. > > What I noticed over the years is that frequently if the initial capturing > step takes too long you will end up with protein that is not necessarily as > functional as you wanted it. You can add cOmplete tablets (if you don't > work on proteases) of course, but sometimes you can't do that and then it > gets important to clean up the sample ASAP. > > I had my protein once almost completely disappear because I thought I > could go quickly shopping while the centrifuge is spinning down the lysate. > So I added an extra hour instead of 30 minutes. Well that purification > ended in nothing. I believe I learnt something - did I ? > > Just a thought > > Jürgen > > > On Aug 22, 2013, at 1:47 PM, Ed Pozharski wrote: > > On Thu, 2013-08-22 at 10:07 -0400, Bosch, Juergen wrote: > > Yes, I'm also surprised why people run gradients for the capturing > > step ? > > > Because we can. Joking aside, I've seen some examples where protein > eluted at relatively low imidazole and upon running the gradient there > remains some (minimal) overlap with non-specific binders. Mainly I just > never seen consensus on what is the right imidazole concentration for > the wash buffer (10mM? 50mM? may even depend on expession > circumstances), running the gradient takes that uncertainty away. Also, > batch method probably leaves more contamination behind. > > One can run imidazole gradient in 1-2 hours, batch is not really much > faster, if at all. So if one has FPLC access, doing imidazole gradient > seems like a good standard policy. Benefits might be minor and rare, > but it is not much more work and certainly not worse than batch in any > respect. > > My two cents (which in Russia turns into three rusty kopecks), > > Ed. > > -- > After much deep and profound brain things inside my head, > I have decided to thank you for bringing peace to our home. > Julian, King of Lemurs > > > ...................... > Jürgen Bosch > Johns Hopkins University > Bloomberg School of Public Health > Department of Biochemistry & Molecular Biology > Johns Hopkins Malaria Research Institute > 615 North Wolfe Street, W8708 > Baltimore, MD 21205 > Office: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-2926 > http://lupo.jhsph.edu > > > > > -- David M. Mueller Biochemistry and Molecular Biology The Chicago Medical School Rosalind Franklin University of Medicine and Science 3333 Green Bay Road North Chicago, IL 60064 *david.muel...@rosalindfranklin.edu http://lmb-il.org/index.html *Office: 847-578-8606 FAX: 847-578-3240
