How to understand N-terminal sequencing data?

Recently i got for N-terminal sequencing of my three different protein samples and got results in form of a table. the sequence predicted is quite different from what we already checked through in...

07 August 2017 9,718 0 View

How to get pure protein from Ni-NTA which gives protein with lot of contaminants?

I am trying to purify one of my protein of interest having molecular weight around 52 Kda. Upon over expression it comes in inclusion body. So I isolated and washed inclusion body and then...

07 August 2017 6,735 10 View

Why i am getting activity in 8M Urea denatured protein?

Actually i am trying to express and purify a microbial metalloprotease. I successfully developed the construct and cloned it in E.coli cells and the protein got expressed in form of inclusion...

06 July 2016 9,093 3 View

Do people wanna write guest article or become a regular community contributor at BenchNEXT?

Hi, My name is vishal and excuse me if this post looks like an endorsement to you. You are free to leave it and ignore. Actually i with my two seniors thought of creating an alternate platform to...

02 March 2016 2,521 0 View

Why is my protein eluting out in wash buffer under denaturing condition?

I am purifying my protein under denaturing condition and washing the inclusion bodies before with washing buffer (50mM Tris, NaCl-500mM, Urea-2M, Triton X-100- 1%, pH-6.8) and Suspension buffer...

11 December 2015 5,017 6 View

How can I stop the protein coming out in flow through while doing purification?

I am trying to purify protein having molecular weight ~50Kda under denaturing condition through Ni-NTA chromatography and got following profile. Any suggestion to stop protein coming in flow...

05 June 2015 6,151 15 View

How do you solve No expression of a protein problem in E.coli?

01 February 2015 7,069 8 View

How can one solve the problem of getting expression bands lower than expected size?

I am working with a protease gene which is his-tagged at c-terminal and is ~50Kda size. I transformed the recombinant construct (pET23b+Gene) in Rosetta DE3 pLysS. When I am trying to check...

31 December 2014 8,998 10 View

What should you do if you get no colonies after transformation in E.coli BL21 de3 cells?

08 September 2014 4,479 8 View

How do I set up a ligation reaction?

I had performed restriction digestion of insert and vector for 3 hours and gel extracted them using a qiagen gel extraction kit in 60 ul of nuclease free water. Thereafter I tried to measure the...

07 August 2014 7,618 11 View

Which is a lucid standard comprehensive book to guide on psychological scale construction and its standardization?

02 March 2021 7,082 3 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 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

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View

Does anyone have a sequence for the human CD34 promoter?

I'm looking for the human CD34 promoter sequence to engineer into a vector construct. Can anyone recommend a good source for this sequence? Thanks very much, Laura

25 February 2021 8,205 1 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View