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. * Stored the resin in 30% etanol. I loaded only recharged bead on the gel to check I could see nothing on the sds-page or not, but I could see faint or visible proteins on sds page due to protein and Ni didnt strip off completely. please let me any solution for it. Thank you Regards Sudheer ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 which will change the pH of the reaction mixture when i increase the concentration of urea. Thank you Regards Sudheer ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
Re: the absorbance value 1.05 at 260/280 of protein solution
Hi all Thank you very much for the replies. On Wed, Oct 28, 2015 at 7:40 PM, Nair, Jayakumar < r.jayaku...@roswellpark.org> wrote: > Nothing if you are measuring only protein concentration. A Lot if you > were measuring DNA concentration. > Jay > > > -Original Message- > 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 > > Hello people > > What does it mean by the absorbance value 1.05 at 260/280 while measuring > the protein concentration at 280 nm ? > > Thank you in advance > -- > Sudheer Babu.S > Research Fellow > Institute of Biochemistry > Biological Research Center > Szeged,Hungary. > ___ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > > This email message may contain legally privileged and/or confidential > information. If you are not the intended recipient(s), or the employee or > agent responsible for the delivery of this message to the intended > recipient(s), you are hereby notified that any disclosure, copying, > distribution, or use of this email message is prohibited. If you have > received this message in error, please notify the sender immediately by > e-mail and delete this email message from your computer. Thank you. -- Sudheer Babu.S Research Fellow Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 same in sensitivity in visualizing the protein bands? Thank you in advance Sudheer ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 didnt show dimer(as expected), only one position(proteinL127pBpa) have shown thickest dimer band rest(each protein has pbpa in various position) shown faint dimer. I suppose not to get faint dimers everywhere where crosslinker available, atleast few should not have shown faint dimer. I am not getting what to do? Could anyone have suggestions to my problem? Cheer Sudheer -- ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 concentration directly. Because I need to handle many samples all the time, so it is difficult for me to do bradford or lowry methods all the time. So How far is correct if I estimate the protein concentration directly? please give me your suggestion Cheers -- Sudheer Babu.S Research Fellow Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 a 1mg/ml solution, but to the same protein in another article they determined the concentration by giving 66350 /m/cm. If i want to check my protein concentration by using nanodrop which value I should concern and how did they get value of 2.7 ??? if anyone has idea please reply me Thanking you in advance Cheers -- Sudheer Babu.S Research Fellow Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 Research Fellow Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 in colour. could anyone explain why it has happened and how I could get back Ni-NTA beads to normal light blue color. Thank you in advance -- Sudheer Babu.S Research Fellow Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 PBS,5000ml at kept at 4 degree for overnight. The next day morning I collected the samples. I changed the dialysed buffer only once, If I do this way the purified protein is free of imidazole and the protein properly soluble in 1xPBS??? I did this way because I dont want use much PBS ? 2. The recombinant protein purification: The protein , which I am purifying most of the protein bind to Ni-NTA though the purification process was finished. I could say 70% protein I am getting in elution fractions remaining protein still stick to the beads. I wanted to reuse the columns for many times. So Could anyone please suggest any buffer, which can wash and elute the protein from the bead So I can reuse the column. If anyone answer me for my questions that would great help to me from them. Thank you very much in advance Cheers Sudheer ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 plate, which I can use for photocrosslinking. Thank you in advance Cheers -- Sudheer Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 µgs more volume to be loaded but I could load maximum16 µl in each well. In that case, should I use C1 V1 = C2 V2 formula, 1000 µg * ? = 16 µg * 8 µl V1 = 0.128 micro liter from the stock, 7.8 ul milli q water, to that add 8 µl 2x sample buffer and load 16 ul to the well, Am I right doing things here? Thank you in advance -- Sudheer Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 500 ng/mico liter. I didnt check the plasmid by running agarose gel. One of my labmate told chormosomal DNA also could have come when I resuspend with pipette. When I did bacterial transformation with this plasmid I got few colonies. Could any please tell, did I do any mistake. Shall I do further steps. -- Sudheer Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
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 that, but not like earlier. After the OD was 0.55, i induced with IPTG but unfortunately i could nt able to express the protein. The only mistake I did was LB media was little diluted, except this, I think I have done everything right... With curiosity, I would like to know what could be the problem? If any one has any idea.. Thanks in advance -- Sudheer Babu.S Ph.D student Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
