[ccp4bb] postdoctoral research associate job

[David Blum]

Apologies for the off-topic post, but the position in my group may be of
interest to students who have skills in protein expression and purification
wanting careers in industry.  Many of my former students, staff and
post-docs have moved to high paying jobs in biotech and biopharma.

The Bioexpression and Fermentation Facility (bff.uga.edu) is a contract
research unit within the
University of Georgia. We are looking for applicants for a postdoctoral
research associate who will
receive advanced training in bioreactor and chromatography operation and
work on fermentation, cell culture and protein purification projects
contracted through the facility. The postdoctoral research associate will
have the following Duties/Responsibilities.

Email CV to David L. Blum, PhD, Director: *b...@uga.edu* 

https://www.ugajobsearch.com/postings/209088

*% Time*

*Duty/Responsibility*

50

Executing fermentation projects.  Includes setup, cultivation and cleaning
of system.  Data collection and analysis

40

Executing cell culture and purification projects.  Requires employee to
understand operation of cell culture equipment.  Understand protein
purification equipment and execute projects as needed.

5

New technology development

3

Train and mentor students (undergraduate and graduate) on equipment and
techniques. Allow shadowing opportunities for students

2

Other duties as assigned



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Re: [ccp4bb] analytical gel filtration columns

[David Blum]

Mohinder,

You could also try HPLC where you should get better results due to more
theoretical plates.  One option would from Phenomenex.
10E6A Phase Information
--

   - Molecular Weight Range: 60 K - 10,000 K

https://www.phenomenex.com/Products/HPLCDetail/phenogel

*David L. Blum, Ph.D.*

*Bioexpression and Fermentation Facility | Director*

*Master of Biomanufacturing and Bioprocessing | Director*

*120 Green Street | Life Sciences Bldg rm A414A | Athens, GA 30602 *

*706-542-1035* <706-542-1035>* | **b...@uga.edu* * | *
*bff.uga.edu* * | **biomanufacturing.uga.edu*





On Wed, Apr 17, 2019 at 11:11 AM Mohinder Pal 
wrote:

> Dear all,
>
> I would like to gel filter a multi protein complex (1.4MDa) as the final
> purification step. I have tried tandem Superose 6 columns and this complex
> elutes close to void volume as it is a very elongated molecule.
>
>
> I was wondering if someone could suggest different analytical columns for
> better resolution as I plan to add more components to this existing
> complex.
>
> Best wishes,
>
> Mohinder Pal
>
> --
> "Whatever you’re meant to do, do it now. The conditions are
> always impossible.”
> Doris Lessing
> --
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] the purification process of protein, used for crystallization

[David Blum]

Hi Liuqing,

I would not recommend SEC.  SEC does not give that great of a separation
unless your contaminant is greatly different in size.  Instead of SEC, you
might want to consider hydrophobic interactions chromatography (HIC).  You
can add your ammonium sulfate directly to your eluted protein from your
IMAC column or IEX, avoiding the dialysis step.  I would also recommend
trying some test kits which have a variety of columns to test and see what
works best for your protein.  We use these kits routinely for our clients
and have good luck with HIC.



*David L. Blum, Ph.D.*
Department of Biochemistry and Molecular Biology | *Director, Bioexpression
& Fermentation Facility*

120 Green Street | Life Sciences Bldg A414A | Athens, GA 30602
706-542-1035 <7065421035> | b...@uga.edu | bff.uga.edu

On Fri, Nov 17, 2017 at 8:44 AM, Liuqing Chen <519198...@163.com> wrote:

> Hello everyone!
> I have listened someone suggested that,  first use affinity chromatography
> (Ni-NTA),  then use SEC (superdex200 increase),  and finally used ion
> exchange (monoQ),   to purified protein,  which will be used to
> crystallization.
> My question  is why  the monoQ used in the finally step,  why not the SEC
> used at the finally step?
>
> sincerely
> Liuqing Chen
>

Re: [ccp4bb] off topic

[David Blum]

Hi Anamika,

I did a search and it looks like researchers are using either mammalian
cells or baculovirus to express this protein.  I don't have experience with
this particular construct so could you tell me why you choose *E. coli*?  I
run a protein expression facility and we typically use HEK or CHO cells and
get mg amounts from cell culture of mammalian derived proteins like STAT1.
Happy to talk offline if that would be easier.

Best,

David

-- 

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

(706) 542-1035 (Office)



On Thu, Jul 6, 2017 at 10:11 AM, Anamika Singh 
wrote:

> Hi,
>
> Is anyone has worked with STAT1 proteins?
>
> I have cloned the SH2 domain of STAT1 protein into pet28a vector but there
> was no expression so far or rather say inconsistent expression. Sometimes
> the expression was in inclusion bodies.  I have tried different methods to
> pull out the protein from inclusion bodies using urea, guanidium chloride
> tween20 but none of them worked well. The yield was very low (very faint
> band on SDS-PAGE ) from 3-liter culture. I changed the host cells from BL21
> to Rosetta DE3 cells but no success so far.
>
> We thought to use some other vector system like with SUMO tag but did not
> proceed because the aim of the project to design inhibitor and tag will
> interfere.
>
> Please suggest me something so that I can complete my project in lesser
> time.
>
>
> Looking forward to valuable suggestions.
>
> Thanks
> Anamika
>

Re: [ccp4bb] off topic: Alternatives to GE HiTrap columns

[David Blum]

We have had good luck with columns from Essential Life Solutions (
http://www.essential-life.net/).  They are easy to work with and hold up
under pressure well.

Best,

David

-- 

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

Skype: dlblum11

(706) 542-1035 (Office)

(706) 542-1077 (Fax)

On Wed, Feb 15, 2017 at 1:04 PM, Carlos Kikuti  wrote:

> Well GE sells empty columns (XK for low back-pressure, tricorn for higher
> back-pressure) that you can fill with the resin you want. They seem
> expensive but for that kind of things I don't think any option will be real
> 'savings'. For nickel affinity we have been having good results with
> Roche's Complete - it requires less imidazole for binding and elution, and
> at the same time it is compatible with reducing agents and chelators. And
> we still buy the HisTrap FF crude.
>
> On Wed, Feb 15, 2017 at 6:03 PM, Christian Roth <
> christianroth...@gmail.com> wrote:
>
>> Hi Markus,
>>
>> BioRad has columns as well (prepacked and empty), which follow the
>> establiched connector standard. So usable without the need of adapters.
>>
>> Cheers
>>
>> Christian
>>
>> Am 15.02.2017 um 16:57 schrieb Markus Seeliger:
>>
>> Dear all,
>> we are happy users of all that GE offers around their FPLC system, but I
>> am getting a little tired of feeling monopolized. Are any of you aware of
>> either empty columns or prepacked columns (e.g. metal affinity or ion
>> exchange resins) from other companies?
>> Thanks for your advice
>>
>> Markus
>>
>>
>>
>

Re: [ccp4bb] Manual His-Tag Purification

[David Blum]

Hi Sundaram,

The binding capacity of this column is 40 mg/mL of resin so a 5mL column
will hold a maximum of 200 mg of protein.  If you run your cleared lysate
on a gel you may be able to estimate how much protein there is.  Our
facility purifies a range of different proteins for investigators and our
rule of thumb is not to load more than 1/3 of the column capacity so if
your construct expresses more than 66.6 mg/L then you may want to batch
load the protein.  Without any knowledge of expression levels, I would
recommend loading 1/10 of your cleared lysate then estimating total protein
from your purified sample before loading the entire lysate.

Best,

David

-- 

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

Skype: dlblum11

(706) 542-1035 (Office)




On Wed, Feb 1, 2017 at 5:08 AM, Tim Gruene  wrote:

> Dear Sundaram S,
>
> during my PhD I used 4-7.5ml resin per l of culture, but I also had a large
> yield of 60-100mg protein per litre. Try to use as little as possible - at
> first trial check both flow-through and retentate by SDS-PAGE. (see. p. 40
> and
> 54 of my thesis).
>
> My constructs would also bind greatly to Ni-IDA, but not at all to the much
> more common Ni-NTA.
>
> When you expect low yields, and a protein that may be sensitive to the
> immidazole concentration, you can also try Co instead of Ni.
>
> Best regards,
> Tim
>
> On Wednesday 01 February 2017 02:54:09 PM Sundaram wrote:
> > Hello ,
> >
> > It's an off topic question.
> >
> > I'm planning to do manual purification a 6 his tagged protein of size
> > around 20kDa from 1L E.coli culture using
> > COHISC-RO Roche cOmplete™ His-Tag Purification Column.
> >
> >
> > Can I get some advice regarding the lysate loading volume and retention
> > time.
> > This is the first time I going to use this column and I have no idea
> about
> > my protein yield from 1L culture.
> >
> > Sorry if I spammed your inbox.
> >
> >
> > Thanks!
> >
> > Yours Sincerely,
> > Sundaram.S
> --
> --
> Paul Scherrer Institut
> Dr. Tim Gruene
> - persoenlich -
> Principal Investigator
> Biology and Chemistry
> OFLC/102
> CH-5232 Villigen PSI
>
> Phone: +41 (0)56 310 5297
>
> GPG Key ID = A46BEE1A
>
>

[ccp4bb]

[David Blum]

Sanjit,

We routinely use Protein G coupled resins for purification of monoclonals
produced in the facility.  On a structural level, antibodies typically
recognize 5 or more residues in a protein unless the immunogen, such as a
single amino acid in your case, is a hapten conjugated to a carrier
molecule.

Could you be more specific regarding your rationale and why conventional
methods (e.g. Protein A or G) are not appropriate for purifying a
monoclonal antibody?

Best,
David
-- 
David L. Blum, Ph.D.
Bioexpression and Fermentation Facility
Department of Biochemistry and Molecular Biology
University of Georgia
120 Green Street room A414A
Athens, GA 30602
http://bff.uga.edu/
(706) 542-1035 (Office)


On Tue, May 13, 2014 at 7:32 AM, Sanjit Roy sanjitkr...@gmail.com wrote:

 Dear All
   I am planning to purify monoclonal antibody.  In this
 concern I found Pseudospecific ligands such as histidine,
 tryptophan,phenylalanine etc. can be used to purify a wide range of
 bio-molecules and especially immunoglobulin. I wish to know the structural
 mode of binding of Histidine (as affinity ligand) with Immunoglobulin.
 Sanjit Kumar

Re: [ccp4bb] project and literature organization software (laboratory information management software)

[David Blum]

Dear Tobias,

My group uses a web based content management system called drupal to manage
inventory, orders and are now setting it up to manage data.  Drupal has a
SQL server to manage different types of information and it is normally
utilized to build websites, but you can also use it as a database only.  To
do this, you make different tables (drupal calls them content types) for
your results project ideas, literature or whatever else you want to manage.
 Since the database is relational you can setup fields that lookup
information from other content types or you can just put in a hyperlink
since it is also a webpage.  You can attach an unlimited number of files,
but we usually put in links to the files that are stored on our server.
 Happy to talk in more detail if you want to contact me offline.

David Blum
Bioexpression and Fermentation Facility
University of Georgia
b...@uga.edu
bff.uga.edu



On Tue, Apr 29, 2014 at 7:21 AM, Tobias Beck tobiasb...@gmail.com wrote:

 Dear all,

 I am looking for a software solution to organize many pieces of information

 1.) Results from (bio)chemical experiments, such as spectral data,
 pictures.

 2.) Project ideas, milestones, etc.

 3.) Literature, including tags, short comments, etc.

 For example, for a certain project I would like to collect information
 about experiments conducted, then link this to literature/literature
 experiments and to project outlines. All this should be accessible for
 multiple users on different OS.

 I have briefly looked into PiMS (too much crystallography oriented),
 Contor ELN (only on Safari on Mac?), Labguru (nice, but not too flexible
 and mostly for biosciences) and Confluence (nice wiki, but so far no real
 literature plugin).

 I know that this sounds maybe a little bit like something called in German
 a 'eierlegende Wollmilchsau'
 http://en.wiktionary.org/wiki/eierlegende_Wollmilchsau 

 But I would be happy to hear about what software people (and labs) have
 tried, liked/disliked and
 ideally the reasons.

 (I am aware that there was a similar query
 https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg24657.html, but
 this was more than 2 years ago)

 Thanks a lot!

 Best wishes, Tobias.

 --
 ___

 Dr. Tobias Beck
 ETH Zurich
 Laboratory of Organic Chemistry
 Vladimir-Prelog-Weg 3, HCI F 322
 8093 Zurich, Switzerland
 phone:  +41 44 632 68 65
 fax:+41 44 632 14 86
 web:  http://www.protein.ethz.ch/people/tobias
 ___

Re: [ccp4bb] TEV protease alternatives and Proteases which leave little on new C-term

[David Blum]

Dear Jacob,
We have been using factor XA for several years for tag removal of the
proteins we express in the facility.  Factor XA does not leave an overhang,
cutting after an IEGR sequence.  It can be quite expensive though so we are
looking into methods to purify it from bovine plasma.

David Blum
Bioexpression and Fermentation Facility
University of Georgia
b...@uga.edu
bff.uga.edu




On Mon, Apr 28, 2014 at 3:01 PM, Keller, Jacob kell...@janelia.hhmi.orgwrote:

 Dear Crystallographers (this may be off-topic, depending on whom you
 ask...)

 A: Can anyone recommend new improved alternatives to the usual suspects
 for proteases (TEV, thrombin, enterokinase, etc.)? I've seen some
 literature about SUMO- and NEDD8-dependent enzymes, but those apparently
 require the whole protein domain to be in the construct for cleavage to
 happen, rather than just a short sequence motif. Further, I'd be curious
 whether there be drawbacks to using the various new breeds that might not
 be mentioned in the original publications thereon.

 B: While I have seen several proteases that leave behind only 1 aa on the
 new n-terminus of the target, I've yet to come across proteases that leave
 behind very few residues on the new c-terminus, which would be very helpful
 for tagging cleanly the c-terminus of proteins. Do such enzymes exist, and
 if so, are there particularly good ones? And if not, I wonder why proteases
 usually require more sequence on the n-terminal side of the scissile bond?

 All the best,

 Jacob Keller

 ***
 Jacob Pearson Keller, PhD
 Looger Lab/HHMI Janelia Farms Research Campus
 19700 Helix Dr, Ashburn, VA 20147
 email: kell...@janelia.hhmi.org
 ***

Re: [ccp4bb] what happens when freezer goes down

[David Blum]

Jackie,

We grow cells routinely and freeze pellets after fermentation.  In general,
proteins are fairly stable until you break cells so you are probably ok
unless your protein is very heat labile and it sat at room temperature for
hours.  However as I mentioned, there is a kind of buffering from the cell
that can stabilize the protein until you are ready to use it.  The glycerol
stocks are probably ok as well since you need just a small inoculum to get
your culture growing.  The plasmids may be the most affected, but you can
sequence if you have aberrant expression.

Best,

David

-- 

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

b...@uga.edu


On Fri, Apr 25, 2014 at 12:43 PM, Jacqueline Vitali jackie.vit...@gmail.com
 wrote:

 Dear colleagues,

 I know this is not related to ccp4 but I am in need of an answer and many
 of you work with cells etc.

 My building had a major malfunction of electricity and the power backup
 did not kick in.  My -80C freezer was without power for over 24 hours until
 I found out.  Because it is small, it goes fast to room temp.  I had many
 glycerol stocks in it with cells, cells with plasmids etc. as well as cell
 pellets.  I am trying to rescue things.

 My question is what happens to cell pellets.  I had many as I like to
 start purification at the cell pellet level.  Are these destroyed when they
 go to room temp for 24 hours or are they ok?

 Thanks.

 Jackie Vitali
 Cleveland State University

Re: [ccp4bb] off-topic: bug busting

[David Blum]

Dear Phoebe,

We have a constant systems homogenizer that we use routinely as a service
for researchers here at UGA. It is really easy to use and gets up to high
pressure (40k psi) so you can lyse plant cells or other difficult to lyse
cell types.  You just pour/pump your resuspended cells, as low as 25mL,
into the inlet, setup parameters (usually once) and hit the start button.
 The lysed cells will exit the outlet line.  We usually have 2 cups to
catch the outlet and we recycle 3-6 times. The unit will automatically
shutoff when it runs out of liquid.  Below is a link to the website for the
company with a video.  For greater than 1L of cells we have a Niro-Soavi
high pressure homogenizer (second link) that I recommend.

http://www.constantsystems.com/products/cell%20disruption%20systems/ts%20series%20cabinet
http://www.nirosoavi.com/products/Ariete_NS2006.asp


On Tue, Feb 4, 2014 at 11:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote:

  Some time ago, there was a nice discussion of cost-effective, wimpy
 protein-friendly ways to break open E. coli.  We're thinking about
 replacing an aging sonicator.  If people have a favorite gizmo, could
 they repeat that advice?
 thank you,
   Phoebe Rice

  ++

 Phoebe A. Rice
 Dept. of Biochemistry  Molecular Biology
 The University of Chicago

 773 834 1723; pr...@uchicago.edu
 http://bmb.bsd.uchicago.edu/Faculty_and_Research/

 http://www.rsc.org/shop/books/2008/9780854042722.asp

[ccp4bb] Opening for an experienced Protein Purification PhD scientist

[David Blum]

The Bioexpression and Fermentation Facility (BFF) within the Department of
Biochemistry and Molecular Biology at the University of Georgia invites
applicants for a non-tenure track position at the Assistant or Associate
Research Scientist level.

Please visit our website (http://bff.uga.edu) for details.

Sincerely,

David L. Blum, Ph.D.
Director, BFF