Stripped and recharged Ni-NTA beads but still getting impurities only with Ni-NTA beads

Hi * Strip of nickel ions by washing with 10 bed volumes of 100 mM EDTA, pH 8. * Wash resin with 10 bed volumes of de-ionized water. * Charge resin with 2 bed volumes of 100 mM NiCl2 metal ion aqueous solution. * Wash resin with 10 bed volumes of de-ionized water to remove unbound metal ions.

what is the milliQ water pH?

HI can someone tell me what is the milliQ water pH? In my working institute I have two departments, I collected milli Q from both departments and checked the pH. I have got pH 4.3 and 5.4, respectively . I donno which one I should believe. Thanks -- Sudheer Babu.S Research Fellow

pH of urea increasing with increasing concentration

Hi 13 M urea is in MQ. When I add 1M, 2M, 3M each, to the protein solution, the pH of the total reaction mixture is increasing with the increasing the concentration of Urea solution. I also prepared urea in 1x PBS, behaving same. Can someone tell me solution or can some suggest strong buffer

Re: the absorbance value 1.05 at 260/280 of protein solution

age- > From: methods-boun...@oat.bio.indiana.edu [mailto: > methods-boun...@oat.bio.indiana.edu] On Behalf Of Sudheer Sangeetham > Sent: Wednesday, October 28, 2015 7:14 AM > To: meth...@oat.bio.indiana.edu > Subject: the absorbance value 1.05 at 260/280 of protein solution >

different molecular weights proteins 23 and 66 kDa?

Hello Different molecular weights proteins 23 kDa and 66 kDa looks similar with band intensity on SDS-PAGE(coomasie staining) does it mean both proteins contain same concentration? -- Sudheer Babu.S Research Fellow Institute of Biochemistry Biological Research Center Szeged,Hungary.

Can I estimate the ' Fluorescent proteins' concentration at 280 nm ?

Hello is it possible to check my fluorescent protein ( mCherry ) concentration as non florescent proteins at 280 nm with molar extinction coefficient value by Nano drop spectrometer ? Thank you Best Regards Sudheer -- Sudheer Babu.S Research Fellow Institute of Biochemistry Biological

How much time takes to sythesis of one ATP molecule

Hello Everyone Glycolysis produces NADH, FADH futher these will enter into oxidative phosphorylation followed by ATP sythesis. How much time require to get one ATP molecule by breaking down of one glucose molecule. Thanks in advance Cheers Sudheer Sudheer Babu.S Research Fellow Institute of

CBB R250 for microscopy

Hi all I have trivial query to ask you that in my lab *Commasie brilliant blue R250(CBB R250) dye for microscopy, sigma* available instead of normal *CBB R 250, sigma*. My query is that shall I use CBB R250 dye for microscopy to visualize the protein bands in 1D electrophoresis. Both dyes have

photocrosslinking

Hello I am working on recombinant protein UVcrosslinkings to determine the dimerisation sites by sitespecific insertion of pbenzoyl phenyl alanine(crosslinker) into protein in E.coli. After induced, purified the protein, done crosslinking and run SDS Page to check the results. Fortunately Wt

protein estimation

Hello all I would like to quantify the protein concentration. I checked the nanodrop manual in that they have given that I can estimate the protein by measuring directly at 280 nm by giving extinction coefficient value without doing bradford or lowry methods. I would like to choose measuring the

protein extinction coefficient

Hello everyone I wanted to check my protein concentration based on its extinction coefficient by using nanodrop rather than doing Bradford or Lowry methods. I was checking the article in one article, The protein concentrations were estimated using the extinction coefficient of 2.70 at 280 nm for

Re: Methods Digest, Vol 90, Issue 2

Hi Thank you for your suggestions. I have one more doubt, how long time I can use each column? Even in qiagen expressionist, they suggested to use 5 times of each column. After that shall I discard the resin or need to recharge it with recharging buffers? thanks in advance -- Sudheer Babu.S

Ni-NTA column turns yellow colour

Hi everyone After elute the protein from Ni-NTA beads I washed the beads with 0.5M NaOH to get rid off imadozole and traces of protein on the bead, but unfortunately the column turns yellow colour? My recombinant protein which fusioned to m-cherry, even after elution the column looks light pink

Doubt regarding protein dialysis and r-protein purification????

Hello Everybody Hope every research is going well. I would like to ask you 2 doubts 1. Regarding Protein dialyis: I want my purified protein in 1xPBS. So after purify the protein,15ml I dialysed the protein against MQ water,5000ml for 1 hour at room temperature then I dialysed the protein in 1x

Re: Methods Digest, Vol 82, Issue 6

Hi After induction time is over, did you check the protein expression on SDS-PAGE ? If you see on PAGE then you can go for sonication... Hope this helps Cheers Sudheer Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary.

Could anyone please suggest me nontreated multiwell plate for photocrosslinking...

Hello all The protein, which i am working on is likely to attach the surface. I wanna do photocrosslinking for detecting protein-protein interactions. I have gone through many company catalogues but I didnt get what exactly I want. Could anyone please suggest me about non treated mictrotiter

is aluminium sulfate added to coommasie staining for increase the sensitivity of proteins?

Hi all I have read one article(http://www.ncbi.nlm.nih.gov/pubmed/2466647), they added aluminium sulfate to commasie staining solution(CBB R250) to increase the sensitivity of proteins. Is anybody tried it ? Thank you in advance -- Sudheer Babu.S Ph.D student Institute of Biochemistry

how to prepare protein sample, if I want to load exact amount on SDS-PAGE

Hello Guys I have a trivial query, I have a protein stock 1000 µg per ml. Now I would like to load on each well 0.25, 0.5, 1, 2, 4, 8, 16 and 32 µgs. For 0.25 µg, 0.25 µl from stock + 0.25 µl 2x sample buffer , total 0.5 µl load on first lane. If I want to load higher amount like 16 µg and 32

is denature plasmid undergo transformation???

Dear Researchmates I isolated plasmid from zymogen plasmid isolation kit. After pellet down the bacterial cells, I added 7x lysis buffer(kit reagent) and by mistake I resuspend the pellet with pipette( suppose I should not resuspend with pipette). But when I measure plasmid concentration it was

Re:protein purification

Hi Actually getting protein with purity depends on various factors. Are you loosing your protein in washing? How much concentration of Imidazole you are using? -- Sudheer Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary.

Hello...Native Page is not polymerising

Hello Could any please tell me why my Native PAGE is not polymerizing. I have referred one article, which i mentioned below and did in similar way and same concentrations but didnt polymerized. Could any one please tell me the reason. Thank you very much in advance Best Regards -- Sudheer

Re: Sterilised LB media getting some medicine smell, while I was growing E.coli culture

Hi Prasad Thank you for the reply, may be your interpretation is right. In your case when strange smells comes, could you able to express the protein? Thank you in advance Sudheer Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary.

Sterilised LB media getting some medicine smell, while I was growing E.coli culture

Hi Guys I am curious to know one thing that, I sterilised the LB media (which was little more diluted). I inoculated the cultures with suitable antibiotics and while I was measuring the OD of bacteria I could smell some medicine was there from the media, I really dont understand why it was like