://www.accelagen.com/Turbo3C.htm) available at a reasonable price. It has
dual GST- and His-tags for easy removal. We have other enzymes for protein
purification.
Chun Luo
Accelagen
From: CCP4 bulletin board On Behalf Of Gloria Borgstahl
Sent: Wednesday, December 7, 2022 12:26 PM
To
Many phosphatases, such as lambda phosphatase, have good soluble expression in
E. coli. Their activity can be shown by simply colorimetric assay.
From: CCP4 bulletin board On Behalf Of P. H
Sent: Wednesday, June 16, 2021 3:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Looking for protein
AcTEV protease is a TEV protease catalytic domain with a stabilizing mutation.
There is a patent on the mutation, although most people ignore the patent.
We have TurboTEV (http://accelagen.com/TurboTEV.htm) which is not stabilized by
the patented mutations. It costs a fraction of the price of
ese mutated
protein and used it in my lab for academic research, does the lab that I
represent have to pay anything, as this is a non profit mailing list?
looking forward to your answer.
Kind regards
André
- Original Message -
From: "Chun Luo"
To: CCP4BB@JISCMAIL.AC.UK
Sent: W
Hi Nicola,
Our TurboTEV (http://accelagen.com/TurboTEV-datasheet.htm) is very stable and
does not need additional reducing agent for its activity. The storage buffer
has 1 mM TCEP. It'll be likely <0.1 mM in your digestion. You can get it from
our European distributors or directly from Accealge
Not sure if this helps.
We ship proteins with dry ice. The dry ice label has lines for Shipper and
Recipient (Consignee) addresses which are often not filled out. We had one
package returned for not filling them out.
Also check your bill. FedEx charge the return.
--Chun
From: C
Hi Chris,
Does you protein polymerize?
We had examples that protein solution turned turbid at high temperature and
become aggregates. We also had proteins precipitated on ice but not at 4C.
Sometimes not much protein loss after clearance.
Looks like your Ni-NTA buffer (500 mM NaCl, 30 mM HEPES
In addition to price, the prevalence of Ni purification may be another reason
for Tris popularity. Some His-tagged constructs don't bind to Ni well in HEPES.
I wonder if anyone has similar experience or comments. --Chun
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL
Hi Giulliana,
You can check our general protocol for TEV digestion
(http://www.accelagen.com/TurboTEV-protocol.htm). Once you mixed TEV with
protein solution, let it sit for digestion. No shaking or rocking. We found
shaking or rocking sometimes reduce the efficiency.
Some constructs jus
Accelagen (http://www.accelagen.com/) a fast growing gene-to-protein company
providing protein expression and purification services. We are seeking a
highly motivated Ph.D scientist to fill a full-time Research Scientist
position for recombinant protein expression and purification. Extensive
experi
Adding TurboNuclease (http://www.accelagen.com/TurboNuclease.htm) to the
lysis buffer can significantly reduce the lysate viscosity especially when
minimum lysis buffer is required. --Chun
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji
Edayathumangalam
Sent: Thursday
Toxicity of target proteins in pET vectors can manifest itself without DE3.
Some people suggest E. coli polymerases causes low level of expression. The
observed mutation rate is thousands of fold higher than what the textbooks
say. Its easy to tell toxicity from other causes of heterogeneity. Less
Make sure the imidazole is removed before you apply the TEV digested sample
back to the Ni column for the reverse Ni step.
Sounds like the expression level of your protein is low. You many consider
to improve the expression. Those contamination proteins disappear from Ni
elution when the target
It's not difficult to replace parts of AKTAprime. But the pump may not be
available anymore. It should be cheaper to get a new AKTAprime plus than
getting a service contract for AKTAprime. AKTAprime plus costs just a little
bit over $10,000 for a new one and lasts at least 5 years without a service
Although the path length of NanoDrop is fixed. 1 ul may not form good liquid
column. One trick is to spot a little bit more sample, 2-4 ul, on Nanodrop,
specially for concentrated protein samples with glycerol and E. coli
culture. It eliminate the bubble problem.
To get good reading, a new drop n
His-tag is close to neutral but flag-tag is acidic.
Using flag-tag for affinity purification usually gives cleaner protein but
it costs a fortune due to low binding capacity of commercially available
anti-flag resins.
--Chun
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Be
Properly made TEV protease should work with or without rocking. We have done QC
of our TurboTEV, a dual GST- and His-tagged TEV, under many conditions and used
on hundreds of proteins. The most common cause of incomplete digestion is the
insolubility of target protein or bad construct design. Sh
As many have mentioned, there is no need to titer baculoviruses for protein
expression purpose. Plaque assay or other titering methods can give >10-fold
variation between two operators. Cells can be different from time to time as
well. Sf9 cells in different labs are quite different. MOI report
Hi Gloria,
Accelagen offers TurboTEV (http://www.accelagen.com/TurboTEV-detail.htm) at
an affordable price. Please contact us for bulk order discount.
TurboTEV has dual GST- and His-tags and has high specific activity. In our
experience, a 1:50 (w:w) ratio cleaves most of the tags. 1:100 ratio wo
Adobe Acrobat Pro should convert any pdf file (text or image) into a format
recognized by text editing program. --Chun
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of James
Stroud
Sent: Wednesday, November 17, 2010 12:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [
For large amount of media, it's actually cheaper to buy liquid media in bags
when factor in the cost of labor and water. MilliQ water may not be low
endotoxin. --Chun
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Nathaniel Clark
Sent: Monday, Jun
Hi Matthew,
TEV is probably the least robust protease among those commonly used for tag
removal. Here’s a common unit definition of TEV. One unit (corresponding to
0.1 ug TurboTEV) cleaves ≥85% of 3 μg control substrate in 1 hour at 30C.
You need to use really a lot of TEV. Information collecte
Hi Sivaraman,
NAD+ has A259 peak absorbance. So you may not have that much nucleic acids
contamination.
However, it is not uncommon to have large amount of nucleic acids eluted
from Ni columns. Adding our TurboNuclease
(http://www.accelagen.com/TurboNuclease-protocol.htm) in lysis buffer ca
NEB recommends adding Zn for Antarctic Phosphatase. Although we found it
works in all NEB restriction enzyme buffers.
The answer can be found at
http://www.neb.com/nebecomm/products/faqproductM0289.asp.
--Chun
_
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf
Look into AKRAprime. It's good for low pressure columns (Max 1MPa) and costs
~$14,000 with accessories. For the price of a FPLC, we have several
AKTAprime for running SEC columns. --Chun
_
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Sangeetha Vedula
Sent: Thur
TurboNuclease digests DNA and RNA to 1-4 base long fragments. It's very
useful to remove nucleic acids during protein purification and virus
purification. Another benefit of using TurboNuclease at cell lysis is to
significantly reduce lysate viscosity. So it reduces the lysate volume for
column loa
ector less useful.
BTW, Accelagen has TurboTEV with dual GST- and His-tags. TurboTEV is
stabilized through a mechanism different from the common S219 mutations. So
it does not infringe the recently issued Yale patent.
Cheers,
Chun
Chun Luo, Ph.D.
Accelagen, Inc.
6044 Cornerstone Court West, Su
Put the His-tag at C-terminus.
Remove rare codons
Optimize sequence for translation
You probably got truncations not degradations.
Chun
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Kn Ly
Sent: Thursday, March 19, 2009 4:53 PM
To: CCP4BB@JISCMAI
Hi Fred,
I doubt His-trap column be any better. I never found much a difference
between these resins or columns. If you want to try, use His-Trap HP, not
His-Trap FF.
Doing batch binding may help you figure out the problem. In some cases,
protein binds better in batch mode. You can take beads out
We have used Nanodrop for several years and found the readings are always
accurate. The highest concentration we have measured is around 30 mg/ml. The
differences between diluted and concentrated samples are within dilution
error. Nanodrop spectra at low concentration are noisier. We actually prefe
Many years ago, someone from UMass told me that he has crystallized many
proteins with GST-tag and believed that dimerization of GST helped
crystallization. I don't remember his name. --Chun
_
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Nathaniel Echols
Sent: Thurs
Kumar,
Sometimes a pGEX vector with thrombin site causes such a problem. Using TEV
or HRV3C sites may help.
Did you see any band corresponding to the protein without GST?
A simple way to check expression is to induce at 37C for 0.5-2 hours and run
a gel of whole cell lysate. If there is
Another option is to purify in denatured condition, then refold. --Chun
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Meg
Sent: Tuesday, September 23, 2008 12:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Troubleshooting protein purification cation
right. I guess you
already have a refolding protocol. Your protein may refold well following
the protocol and many host cell proteins don't. You may get rid of many
cellular proteins just by doing the refolding.
Good luck!
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sor
fortune to get
enough to see. They are Ni-NTA linked dyes as well.
If your protein expresses well, you can see it while you growing the
culture.
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road, Suite 107
San Diego, CA 92121
TeL: 858-350-8085 ext 111
Fa
is is due to the
enzymatic activity of your protein. We actually had a case that making an
inactive mutant solved the problem.
Good luck!
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road, Suite 107
San Diego, CA 92121
TeL: 858-350-8085 ext 111
Fax: 858-350
accumulates the
contaminants.
Using AA adds cost. Better to make sure operators clean their gloves with
ethanol.
Very interesting picture. It could be a PhD thesis to figure out what it is.
Good luck!
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road, Suite 107
San
ET vectors with N-His
also have C-His. You may also consider HRV3C or TEV. Not because Accelagen
sells those proteases, but they are more specific than proteases like
thrombin and easier to remove.
Cheers,
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road,
, claiming slow on rate. That's a myth. In our hands, glutathione
resins binds as fast as Ni resins.
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road, Suite 107
San Diego, CA 92121
TeL: 858-350-8085 ext 111
Fax: 858-350-8001
[EMAIL PROTECTED]
www.accelage
azole stock needs to be pH adjusted to the same pH
as the buffer. No matter how strong a buffer is, at the concentration
(25-100 mM) most people use, one should not expect a buffer can buffer
imidazole in the Ni elution buffer, since imidazole is a very strong buffer
itself.
Cheers,
Chun Luo, Ph.D.
JJ
Dilute your fractions for this prep.
Do gradient elution in the future. Again dilute the fractions immediately.
Figure out a better buffer will be helpful.
Cheers,
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road, Suite 107
San Diego, CA
g affinity tag from insect cells at an expression level of ~1
mg/L. From E. coli, a 3-step purification is generally sufficient to purify
protein without using affinity tag.
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road, Suite 107
San Diego, CA 92121
even
detrimental to expression.
Best,
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road, Suite 107
San Diego, CA 92121
TeL: 858-350-8085 ext 111
Fax: 858-350-8001
<mailto:[EMAIL PROTECTED]> [EMAIL PROTECTED]
www.accelagen.com
. Currently technology allows you to scale up to 100s L in an
additional couple weeks. The material cost is actually similar to E. coli
expression. It's faster than trying to figure out how to solubilize a
protein.
Good luck.
Chun
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
that phosphorylations of
over-expressed proteins in E. coli are always auto-phosphorylation.
We have a publication in Protein Expr Purif. 2005 Dec; 44(2):121-9 that
investigated the phosphorylation sites.
Cheers,
Chun Luo, Ph.D.
The Protein Expert
Accelagen, Inc.
11585 Sorrento Valley Road
Many brands of pH paper give 0.5-1 pH reading lower for Hepes buffer. pH
meter is more reliable to measure the pH of Hepes buffers. Tris pH increases
in cold. If TCEP is used as reducing agent, make sure the stock solution has
been pH-adjusted. --Chun
-Original Message-
From: CCP4 bulletin
Try batch binding. Sometimes batch binding for 30 min is more efficient than
column binding. If your protein binds DNA, treat with DNase or Benzonase.
Adding detergent may help as has been suggested. --Chun
_
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
changrui lu
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