Does denaturing conditions alter the protein fluorescence?

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Is there any particular site to insert the fragment in histag vector for further protein purification?

Do we need to place the insert in a specific site in his tagg vector for further protein purification or can we just use any restriction site present in the vector?

08 September 2019 8,236 3 View

Why do i find another protein size on my gel?

I've cloned a gene which size was around 800bp in pet 32 vector next to n-terminal histagg to further purify the protein. when I expressed the protein there was a big band around 50 kda + my...

08 September 2019 6,492 5 View

Why has the protein loss it's red color?

i'm doing a protein purification that i incorporated with mcherry because i want to visualize that after purification.i've tried low temperature 14 degree as well as 37 degree but the protein and...

08 September 2019 3,682 4 View

Wher can be the issue if the cloning doesn't work?

I"ve been doing a cloning about 4 months already. i've done all the controls and I 've used differents vectors and also I've done vector dephosphorylation though I used double restrictions...

05 June 2019 4,240 12 View

Does anyone know a vector which can express protein easily?

does anyone know an histagg vector that I can use to clone a gene easily and further express my gene protein ?

05 June 2019 9,150 3 View

Is there any diifference between self ligated vector and fragment-vector ligated?

after we have done transformation ,is it possible to compare colonies on plates, i mean is it possible to find that fragment-vector ligated are bigger than self-vector ligated?

03 April 2019 2,593 3 View

What is the importance of iptg for protein expression?

sometimes the protein can be expressed even without adding iptg, so is it necessarily to add iptg for protein expression?

03 April 2019 7,377 2 View

Is it possible to do the insert digestion in two steps?

I'm trying to clone my insert in pet 16b with nde1 and xho1 restrictions enzymes but I'm always getting only self ligated vector ,and the vector control also have many colonies than the...

03 April 2019 9,354 3 View

how can i clone my gene in pet 16b cloning for further protein purification?

i'm trying to clone my gene in pet16b to further make histagg protein purification. Where can I do the cloning for a successful cloning and which restriction enzymes to use? I also want to get a...

02 March 2019 6,140 6 View

Can i use these enzymes to digest my vector and fragments and use the same to test the ligation efficiency?

my primers are F:GCATCATATGatgaatgggaccgcaaaccc and R:GCATCTCGAGttagtctttctggtaaggccg,my fragment that I want to clone start by atgaatggga.......,and my vector is pet16b.i used ndeI and XhoI...

02 March 2019 4,088 9 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

Is There Any Feasible Method To Test Fluorescent Compounds Other Than Fluorescence Spectrometers ??

Is There Any Feasible Method To Test The Efficiency Of Fluorescent Compounds Other Than UV Spectrometers ? Suggestions Would Be Appreciated !

02 March 2021 5,785 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

Which fluorescent dye to use for measuring total ROS production?

Is DCFDA appropriate for measuring total ROS production, or can it only detect certain types of ROS? If I want to measure total ROS production, which dye should I use?

01 March 2021 9,447 3 View

How to accurately quantify chitin?

Hi all, I have been working on the quantification of chitin in insect samples and feed for a while but I can notice that the quantification methods (e.g. exposure to HCl, NaOH, and weighing sample...

01 March 2021 1,843 2 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 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

Why does intensity drops as concentration increases?

Is there any reason for decrease in fluorescence emission intensity of HSA,BSA when increase the concentration ?

28 February 2021 3,653 4 View

What's the concentration recommend for recombinant protein use in western blotting?

I'm currently working having problem with the concentration for recombinant protein use in western blot, the one been used is Recombinant Human CD19 Fc Chimera Protein-rndsystem which does not...

24 February 2021 8,514 3 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