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I go with miniprep + sequencing. If you eventually have to sequence a construct, I figure I might as well do it at the beginning. I usually pick 6 colonies and send them for sequencing.
I used to screen everything by colony PCR, and false positives can be a problem. To me, I choose to do RE digests last, its just time consuming.
#153120 Screening for ligation and transformation result
Posted
on 28 March 2013 - 10:06 AM
#152605 How to improve enzyme activity?
Posted
on 20 March 2013 - 09:34 AM
You could try purifying your enzymes with purification tags. Is there any specific reason you are using total protein extract? It is extra work, but optimization is much easier once you have purified enzymes.
If you are already using pET vectors, you could clone into the MCS and use their purification tags.
The temperature is important, but I think the most important part is the cell lysis. Proper cell lysis goes a long way in maximizing the activity and purity of an enzyme.
If you are already using pET vectors, you could clone into the MCS and use their purification tags.
The temperature is important, but I think the most important part is the cell lysis. Proper cell lysis goes a long way in maximizing the activity and purity of an enzyme.
#148178 Protein purification problems: Presence of contaminated proteins along with the
Posted
on 16 January 2013 - 11:01 AM
In my opinion, the elute2-2nd flow looks pretty good. From my experiences, the lower band you are seeing might be very difficult to remove and is somewhat ordinary. You do seem to have a conundrum since 100mM is starting to elute your protein of interest but not all of the contaminants. I recommend changing elute 1 to 125-150mM imidazole and think of it as more of a wash. Then elute with 300mM imidazole and see if the first flow through is cleaner.
The bands you have though are very nice and when I was reading your post it made it sound like you had TONS of contaminant bands, but in reality those are pretty clean fractions. If it is available to you, size-exclusion chromatography would clean these samples up very easily.
The bands you have though are very nice and when I was reading your post it made it sound like you had TONS of contaminant bands, but in reality those are pretty clean fractions. If it is available to you, size-exclusion chromatography would clean these samples up very easily.
#148010 Protein Expression vector with small tag
Posted
on 14 January 2013 - 09:00 AM
Why don't you use a cleavable His tag? That way you have no tag at all after purification/cleavage. look into the pET vectors if you are interested in that.
#148005 Protein precipitated while kept at 4C over night after elution from a Ni-NTA col
Posted
on 14 January 2013 - 07:58 AM
It should be fine. I make sure that I put my proteins through dialysis immediately after I elute them from the column. I have also had this problem when I left proteins in the imidazole overnight. At this point, though you cant really do more than what you already have. The proteins should be OK, but if you can check their activity in any way, that will be the true test.
#147342 GST- tagged protein expressed in insoluble
Posted
on 02 January 2013 - 09:10 AM
You could try a cleavable Thioredoxin (Trx) tag. You just clone your gene in frame with the tag and you can cleave it off later. The Trx tag is supposed to be pretty good for increasing solubility of unstable proteins. We use pET32a-c vectors that contain Trx, S, His6 tags that are all cleavable.
#133901 How to get pure protein of a gene
Posted
on 02 May 2012 - 08:01 AM
extra chunk? I assume you ran your purified protein on a gel and you are getting multiple bands. The best way to get the protein that you want from this mixture is size exclusion by FPLC. See if you have a column available and try that.
