How can I use AKTA protein purifier and UNICORN software ?

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Does Sac1 phosphatase act in cis or trans?

Sac1 phosphatase (Sac1p) exists in ER and golgi. It undergoes this reaction : PI(4)P -> PIP. Certain authors said that Sac1p acty in cis in ER and others said that Sac1p is in ER but act in trans...

05 June 2014 7,972 1 View

What is the function of the His87-Lys55 binding in wild-type mitoNEET ?

In the paper "Mutation of the His ligand in mitoNEET stabilizes the 2Fe-2S cluster despite conformational heterogeneity in the ligand environment" by Conlan Andrea R (2011) published in Acta...

01 February 2014 7,754 1 View

Where are the mutated genes in C41(DE3) and C43(DE3) strains of E.coli ?

C41(DE3) and C43(DE3) strains derived of BL21(DE3) strain E.coli. I am searching for where the mutated genes are in C41 and C43 strains and also the function of these genes in the wild type BL21...

31 December 2013 6,819 3 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

How to replicate the well conditions of a crystal hit with 20%(w/v) isopropanol?

I had a crystal hit in a well with 0.1M HEPEs pH 7.5, 10%(w/v) PEG 4000, and 20%(w/v) isopropanol. Why is isopropanol in w/v here if it's just solvent? How do I recreate this well condition?

25 February 2021 9,761 1 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View

What urea buffer can i use for biotagged protein extraction from streptavidin beads?

Hi all, I have been doing research on biotagged LDB1 proteins in Mel cells. I purify the proteins using M280 streptavidin beads. The yield is very low so I've been trying to optimize it by...

24 February 2021 5,749 3 View

Can I use a protein-devoid condition as my negative control in ELISA?

Hello, I am running a sandwich-type ELISA where I am trying to inhibit a protein-protein interaction using small molecules. For each experiment, I determine my Z' (Z-prime) to determine the...

23 February 2021 7,429 1 View

Is there a way to extract total RNA and protein from cell culture at the same time, without denaturing protein?

Hi all, Is there a way to extract RNA and protein from the same cell culture sample? Because we need to use the protein for gelatin zymography at later time, we are looking for a method that can...

22 February 2021 5,341 5 View

Prasad P.

Hello Mr. Adrien Tereygeol. pls find the attachment regarding akta pilot.

Nishant Kumar Varshney

There are already installed methods in UNICORN software..You can always create new programs using Wizards.Otherwise you can always run manual mode...There are lot of manuals and instructions on GE Amersham website.

Veronika Chromikova

According to my opinion, that software is absolutely not user-friendly :/ . Manuals online are at least some help, but usually simplest and fastest solution would be having someone who knows the system show you/ help you for the first time. Isn't there someone like that at your institute?

Jesús Oria-Hernández

You can start using the Akta equipment in MANUAL MODE; if you have basic chromatographyc knowledge is not difficult to understand it. After initializing the Akta and the Unicorn software go to the SYSTEM CONTROL Window, then to the MANUAL menu and press option 1 PUMP, this display the SYSTEM PUMP INSTRUCTIONS WINDOW from where you can control all the basic functions of the equipment. From the left menu of the window you can select FLOW to set the flowrate, GRADIENT to set length and the percent of buffer B to perform a gradient. In this window you can control the FRACTION COLLECTOR, the INJECTOR VALVE too, etc.