How can I remove C-terminal His tag from pET22b express protein and thrombin from N-terminal?

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How to quantify the fluorescence intensity of only the merge area of an image using ImageJ software?

Dear All: I have to quantify the fluorescence intensity of cells that are fluorescent to two different markers, so i'm interested in finding out if this is possible. Previously I have...

22 July 2024 8,770 1 View

How can I avoid uneven IHC staining on free floating tissue from frozen human CNS sections (cryostat 50um thickness)?

Hello, I've been doing free floating IHC using a VAChT antibody with a 1:1k dilution (139 103 - Synaptic Systems). However, there has been a struggle to keep an even stain with my samples of...

06 July 2024 4,521 2 View

What are these weird structures that appear in my human intestinal organoid cultures?

I'm working with intestinal organoids derived from children's polyps. I use Matrigel and Human IntestiCult growing medium, and sometimes I find these structures that resemble extracellular matrix...

23 June 2024 3,938 2 View

Why don't my intestinal organoids from children's polyps grow?

Hi! I am having trouble growing intestinal organoids from children's polyps. Sometimes they don't grow at all, and other times they look dark and unhealthy, as shown in the attached pictures...

18 June 2024 4,781 3 View

¿Que enfoque metodológico me recomiendan para abordar los gremios tróficos en un área natural de BST?

Me interesa investigar la distribución espacial de los gremios tróficos de aves en un área natural específica de bosque seco tropical, analizando la abundancia relativa de cada gremio en las...

12 June 2024 9,313 1 View

Can you help me with some problems I am having with dsRNA production?

The desired gene region was amplified by PCR and the resulting PCR products were cleaned. Bands were observed in an agarose gel and measured in a spectrophotometer. Then, transcription was...

22 May 2024 6,801 1 View

I am working with silver nanoparticles, by mixing NaBH4 with AGNO3, but it doesn't change color. Why?

The as the solution were mixed together, there was no color change, and then the UV-vis was read every 10min but barely any absorbance was observed. The solutions were 2,1mM of NaBH4 and 0,07g...

20 May 2024 615 1 View

Do you recommend any transportation software for simulating crowd-based transportation systems like crowdshipping?

I've been looking for software that simulates the integration of passenger and freight transportation systems. For instance, tools like Vissim and Visum primarily focus on modeling passenger...

14 May 2024 9,283 2 View

Could anyone please indicate papers with EnergyPlus validated with field measurements in Phoenix, AZ?

So far, I have only found Dissertations.

11 April 2024 2,188 1 View

Are there any expert in dendritics cells derivated from somebody blood monocytes?

I am interested in the study of dendritic cells response under infection with mutant strain candidate to vaccine against TB. But I am begginer in the work with dendritic cells, somebody can help...

20 March 2024 1,587 7 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

A positively charged and 10x his-tagged protein that doesn't bind to any chromatographic resin?

Hello everyone, I am currently working on a protein that is 10x his-tagged and positively charged (predicted pI=10.16). But when I tried to use Ni-NTA column to purify the protein, it's not...

24 July 2024 7,293 4 View

Recommendations for his-tag magnetic beads / resins?

Good day everyone, Trying to perform protein purification for the first time, gotta buy the whole equipment/reagents. Protein of interest 48 kDa (Antithrombin), expressed from his-tagged plasmid...

16 July 2024 3,692 6 View

Can I detect the FLAG sequence of my AAV vector by qPCR?

I am treating human cells with an AAV vector encoding for my gene of interest tagged by FLAG. I want to quantify the mRNA expression of my gene of interest via qPCR to discriminate the endogenous...

14 July 2024 8,482 3 View

The criterion about maximally localized wannier function?

I heard that as the value of "num_iter(tag in wannier 90)" is higher, spread of Wannier function(=WF) is gradually lower in wannier 90. If so, is this procedure that minimize the spread of WF...

13 July 2024 4,608 0 View

What should I keep in mind when designing a gene construct when trying to co-transform to increase protein expression?

I am having trouble expressing a protein and have heard that co-transforming it can improve stability and increase expression, so I am looking to try this experiment. I only need the kinase domain...

08 July 2024 2,935 5 View

Is the wash in my IP the problem or something else?

The image shows 2 western blots. The first one is of protein A, and the second is for the flag tag that the protein is bound to. The lanes are as follows Ladder, Beads, SAB(unbound), and elution....

01 July 2024 3,183 0 View

fluorescent imaging without fixatives?

How to store SDS-PAGE for fluorescent imaging without fixatives? I am running protein samples on SDS-PAGE that are tagged with fluorophores. If I place the gel in a destain solution of...

29 June 2024 8,720 1 View

Why does my protein bind poorly to the Ni sepharose beads?

I am purifying RNAse E in E. coli (118 kDa). I cloned the rne coding sequence on pET28a with a His tag at the N-terminus. The protein overexpressed fine (see attached figures). The binding buffer...

24 June 2024 6,287 4 View

How to predict Protein structure and choose N or C terminal tagging?

Hi, may I know I want to fuse my protein with affinity tag for protein purification in Expi293, how can I determine and visualise how does a N or C terminal tag affect my proteins (structure,...

24 June 2024 8,622 6 View

Amelie Skopp

You can add an endoprotease-cleavage site between your tags and protein of interest such as the tobacco etch virus (TEV) protease site. Then you can even keep our protease e.g. His6-tagged and thus keep it separate from your protein of interest when using affinity chromatography.

Lisandra Herrera Belén

Thank you very much