How can I remove DNAk from my protein solution?

More David Vizarraga's questions See All

How could I stop the autoproteolysis process?

Hi all My name is David Vizarraga. I am trying to purify a protein. But When i purify it, after a few days It begins to autoproteolyze. It forms the same fragments in all bach of protein...

07 August 2017 1,308 6 View

What do you think it is?

Good afternoon. I attach an imagen and my question is what do you think it is? I think It is a lot of microcrystalls. In the top of the drop there are microcrystalls alone (unit) and in the...

03 April 2017 7,024 1 View

A rare purification How could I obtain the monomer or one of the oligomers?

Hi all. I am a PhD student who work in protein purification and crystallization. A few months ago I started with a protein which molecular weight is very big around 100kDa (monomer). When I...

31 December 2016 6,415 2 View

What can i do so my protein will be finished the folding?

Hi all. I have a pure protein but in SDS-PAGE gel there is DNAk in the solution too ( It is together with my protein). In Superose6 column I have 3 proximate peaks which I cant separated. I think...

31 December 2016 3,225 8 View

Protocol for E.coli expression/induction with a Heat shock step?

Hi all. I want to high the amount of the Heat shock chaperones concentration. I read one option is to do a Heat shock step before the protein induction. Anyone know How could I do? Thank you.

31 December 2016 7,073 2 View

What Could I do for improve on the protein expression for end the fold of my protein ?

Hi all. I wrote some times here about a unfold protein topic. The case is I have a pure protein but It is together with DNAk and In the exclusión molecular column I have several peaks that I...

31 December 2016 8,495 5 View

Could My protein be affected with the sonication method?

Hi all. I want purify a protein. The special of my protein is that the oligomers or the aggregates broken when I centrifugated. So I want know If My protein could be affected with the...

11 December 2016 4,641 12 View

Which is the perfect buffer for my protein?

Hi all I want to purify a protein (Pi:4,0) and I am use a Typical buffer (50mM Tris pH: 7,4, 200mM NaCl and 10mM glucose ( decreased the aggregates and It is better for the crystallization than...

10 November 2016 9,685 1 View

Why after centrifugation at 13,2RPM during 2 hours does my protein broken in the linear monormer?

Hello. My name is David Vizarraga and I am a PhD student. I am trying to purify a protein but I always have a oligomer mix in the solution and I can't crystallize it (because I need a pure...

10 November 2016 5,210 3 View

My protein forms aggregates but I couldnt use additives like glucose or glycerol because It forms oligomers What could I do in my purification step?

Hi all. My protein forms aggregates but I couldnt use any additives like glucose or glycerol because It forms oligomers. Thank you

10 November 2016 2,799 9 View

Can I use a HisTRAP column for affinity chromatography?

I'm working on selecting antibodies against a recombinant protein that has a His-tag. My idea is to first bind the recombinant protein to a HisTRAP column and then use this column for an affinity...

07 August 2024 505 3 View

Why does my protein refolded to beta sheet during thermal denaturation analysis?

Hi! So i attempted to understand a novel protein behavior towards heat application by analyzing its secondary structure change. I subjected the protein to a thermal denaturation analysis using...

06 August 2024 1,989 3 View

Low-yield gel extraction problem?

I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel...

05 August 2024 9,798 3 View

What is the best blank for nanodrop if I want to read a recombinant protein concentration?

Is it the "elution buffer" or the "dialysis buffer"? Note: I'll be using NanoDrop OneC

01 August 2024 967 3 View

Transfection in HEK293T cells?

Dear All, I am trying to transfect a pCDNA3.1 vector containing my gene of interest. The purpose is to figure out the localization of the protein of interest. I have fused the protein with GFP on...

31 July 2024 9,892 4 View

How to retain the a GFP tagged gene expression in stable cell line?

Hello , I established a stable cell line expressing GFP tagged to a centrosomal gene having G418 drug selection marker. I validated the stable line by IFA and Western blotting, results are fine....

29 July 2024 5,007 0 View

What is the relationship between protein structure and N or C terminal tagging choosing?

I want to do 2,3-butanediol dehydrogenase(BDH) enzyme purification to confirm its activity for 2,3-butanediol. Before that, I need to confirm which N or C terminal tagging is better for enzyme...

28 July 2024 366 3 View

Why cannot i find my protein on cell surface after antibiotic selection of expressing plasmid?

I cannot confirm cell surface protein expression by flow cytometry even after transfection and antibiotic selection of cells. Does it take long for proteins to express on the cell surface? the...

28 July 2024 3,178 3 View

How does the Retention time effect the Biogas Production?

List down the various factors responsible and how does this effect the overall biogas production when it is related to Hydraulic retention time of the digester based on the feedstock?

27 July 2024 4,800 4 View

Problems with ion exchange chromatography?

I'm having problems with ion exchange chromatography. After applying my sample (a fluorescent protein), a preferential path inevitably forms. The matrix I use is DEAE-Sephacel. Could someone...

27 July 2024 6,605 0 View

Mohammed Milhim

If you add ATP to your washing buffer during purification you will be able to release the protein from the Dnak.

Your protein will not be affecting because it is already folded.

Dominique Liger

Hi there,

DnaK/ADP exhibit the highest affinity towards protein. By proposing ATP in large excess, exchange ADP/ATP should occur and this exchange will result in a weakened DnaK/protein interaction.

David Vizarraga

I read that MgATP mM in a wash buffer It is enough for separated DNAk from my protein