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 anyone have pRuW4inh1 harboring the Erwinia chrysanthemi secretion system?

02 March 2016 7,919 0 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

What degree of missing data in a particular case/participant results in Expectation-Maximisation no longer working?

I'm dealing with a mediation model and am using the PROCESS module in SPSS. Due to SPSS and PROCESS being limited in the imputation methods - being unable to handle multiple imputation - the other...

02 March 2021 4,362 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

How can I represent SDS-PAGE results for machine learning analysis?

I am trying to classify and analyze the results of an SDS-PAGE based array for bacterial detection using machine learning, but I have trouble finding the best way to represent the results with...

27 February 2021 9,176 3 View

Mixed ANOVA - help?

I'm currently working on a piece which asks to conduct an ANOVA - looking at the effect of sporting group (2 levels), and exercise trials (two trials with six levels). I've computed this all...

27 February 2021 9,626 4 View

Why my agarose gel staining sometimes looks not homogenic but like gradient (see the figure)?

I am worried about this overexposure of the upper part of the gel in the picture. Is it possible that this is the result of too much concentration of ethidium bromide? Why is there such a big...

25 February 2021 8,140 3 View

How to Interpret Partial Eta Squared Numbers For a Repeated Measures Anova?

I have obtained Partial Eta Squared Numbers through Two-Way Mixed Anova Analysis. My results for different constructs are: .105, .135, .038, .068, .061 respectively. I wanted to know, whether...

24 February 2021 7,122 1 View

EMSA troubleshooting. Protein-DNA complex not migrating, cold probe lane with multiple bands. Can anyone help?

I'm running an EMSA to show the DNA binding activity of recombinant proteins versus the Wild type. Previous assays run by my research group using a different test system show the protein-DNA...

24 February 2021 8,325 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

How to manage western blot for proteomics data validation?

Hi Everyone, I am a bigginer in western blotting and I am trying to use it for validation of my proteomics data. I have: two regulated proteins with molecular mass of 40 and 73 kDa one control...

23 February 2021 6,671 4 View

Alginate and silk fibroin hydrogels toxic to cells?

Hello, I asm currently working with alginate and silk fibroin hydrogels. I prepared a blend of alginate and aqueous silk fibroin (straight from dialysis) and resorted to ionic crosslinking to...

23 February 2021 8,249 2 View