Protein Purification Techniques

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Questions related to Protein Purification Techniques

How to purify the Bacterial DNA from protein contamination?

I have isolated the genomic DNA from actinomycetes by phenol: chloroform method but i am facing the protein contamination in DNA sample. How to purify the DNA from protein contamination without...

10 October 2016 1,532 3 View

DNA extraction in Euphorbia milii?

The samples we have are from leaves but dried completely at dark and in closed room. We have followed several extraction steps that included kit, kit with PVP and CTAB. However, the thing that we...

18 August 2016 9,213 2 View

Is it possible to do a TCA precipitation after blotting on a nitrocellulose membrane?

I am doing clear-native PAGE and need to do a TCA precipitation to be able to detect my protein of interest (hopefully present in a complex) with the corresponding antibody. I tried to incubate...

08 August 2016 6,809 1 View

Unfolding and refolding -cytoplasmic protein ?

I used different lysis buffer for cytoplasmic protein. when I use higher concentration of GdnHcl, protein gets soluble. Is this correct to do unfolding and refolding for cytoplamic protein.

08 August 2016 2,366 2 View

DNA extraction in Euphorbia milii?

The samples we have are from leaves but dried completely at dark and in closed room. We have followed several extraction steps that included kit, kit with PVP and CTAB. However, the thing that we...

08 August 2016 429 2 View

How can old or reused western blot antibodies cause more variability in the normalized fold change data between blots?

I have been running many western blots and have not been able to always use fresh primary antibodies each use because of a limited supply. The proteins were isolated from cells treated with...

08 August 2016 3,714 3 View

Question about (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES and Triethanolamine(TEOA)?

I find some cell lysis buffer (like 1%NP-40 lysis buffer) has buffer agent in it,the commonly used buffer agent is HEPES or TEOA. Nowdays,I need to get the cell lysis to do the CUAAC...

08 August 2016 4,106 2 View

During protease purification by ammonium sulphte casein used as the substrate is getting precipitated,can you pls suggest a method to remove it?

casein dissolved in medium below boiling point for the purpose of optimization. But it precipitated with ammonium salt. Plz help me to resolve the problem?

08 August 2016 6,564 5 View

Can anyone help with the result of determination of sonication efficiency in my ChIP assay?

In my ChIP assay, the sonication conditions used to generate fragmented DNA are as follows: 100 Watt, 20 Hz, 20s on, 60s off, 15 cycles. After purification, samples are visualized on a 1% agarose...

08 August 2016 7,645 10 View

Why does it have smear from the top of gel?

I have to extract DNA from lung ffpe samples with pure link invitrogen kit and high pure PCR template preparation kit Roche. I use 20 ul 20mg/ul RNase A for each sample (about 50 mg tissues).The...

08 August 2016 8,207 5 View

Does anyone knows if this is possible use the two hybrid system to produce functional recombinant proteins?

Hi everyone: In our lab we are currently working with two hybrid system to study interaction between two known proteins. The following question is to know if the protein of interest, the bait,...

08 August 2016 8,405 2 View

Protein desolubilized by crosslinking?

Hi, all! I am currently trying to do RNA Immunoprecipitation. I am using Arabidopsis root as materials and use 0.5%PFA in PBS and fix for 30min and use Glycine for quenching(20min). After that, I...

08 August 2016 7,940 3 View

How much expression of VSV-G-YFP in 293 cells should I expect to see during amplification of adenovirus?

I'm attempting to amplify Adenovirus containing VSV-G-YFP. After infection with viral stock, I don't see much YFP, but I do observe CPE. I haven't finished the purification to test, but is this an...

08 August 2016 3,422 0 View

Can someone suggest me to refold and purify the extracellular domain of a membrane protein?

I am trying to purify the extracellular domain of a membrane protein (an immunoglobulin fold) by over-expressing it in E. coli. The protein always expressed as inclusion body despite of several...

08 August 2016 2,764 7 View

Protein expression in E.coli, located in inclusion-body, how to purify without losing an enzymatic activity?

I constructed a pET-53 to express yeast gene in E. coli BL21(DE3). The culture condition was culture the BL21(DE3) until OD600 = 0.4

08 August 2016 852 9 View

Is it possible to add some tag proteins to target protein of small molecular weight to acquire the structure by Cryo-EM?

I have a soluble protein of 106KDa which the molecular size just lies on the low limit of Cryo-EM tech. I want to use Cryo-EM tech to acquire its structure. I want to know if I can add the tag...

08 August 2016 248 4 View

Am I washing off specifically bound DNA from my Dynabead/Ab ?

I've been having trouble getting results from ChIP, using Dynabeads Protein G. I used 1x PBS solution to wash the bound bead/Ab/DNA complex 6 times and saved each wash in a separate tube for...

08 August 2016 4,777 6 View

White cloudy precipitate during immunoprecipitation?

I'm doing an immunoprecipitation for a epitope-tagged protein expressed in mammalian cells (DH82- a dog cell line). I re-suspend my frozen cell pellet in lysis buffer (Tris-based with 100mM NaCl,...

07 July 2016 1,813 1 View

What is the difference between technical versus biological replicates in mass spectrometry ?

I have one bulk soil sample which i will extract by two different methods. Both methods have around 6-7 steps before the purified protein is ready for mass spectrometery identification of the...

07 July 2016 4,953 6 View

Does any one have extreme knowledge in purification method of a bio-molecule? I need very precise and gentle method without any harse step?

I need to isolate an enzyme from plasma of blood. The systemic circulation might have some kind of house keeping sub-molecules to maintain the structural and biochemical property of an enzyme. So...

07 July 2016 3,383 6 View

What could be the reasons for smearing of bands in Tris tricine gel?

While running Tris -Tricine gel for my protein. I get bands going fussy as in the uploaded pics. Can some one suggest me how can I troubleshoot this problem?

07 July 2016 6,527 7 View

Does anyone ever had different bands for beta-actin and GAPDH between different cell lines for the same amount of protein?

Hi, I'm having a problem with the loading control of my WB where I'm comparing different cell lines. In the picture I have beta-actin on the top and GAPDH on the bottom and I'm comparing Jurkat,...

07 July 2016 7,770 4 View

How to solve the problem when crystallize proteins which precipitate immediately after purification?

I can get the protein sample,but when I screening the protein to crystallized ,the protein precipitate immediately .I think may be the temperature too high(16。C),so screening the protein at...

07 July 2016 5,467 5 View

How can i get rid off non specific bands in pcr reaction for CDNA?

Hello, every one if i have the following result how can get rid off nonspecific band if you know it is appear only with 2 tested sample as in lane 3,4 of my cDNA , but not appear with control...

07 July 2016 1,501 6 View

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