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akhatr hussain
Mar 7, 2013, 4:40:34 AM3/7/13
to met...@magpie.bio.indiana.edu
Dear sir/madam
I have extracted protein the kidney of mice with RIPA Buffer. Now I want to do the protein concentration estimation of these samples. But my sample is dissolced in RIPA buffer and this does not work accurate with bradford assay as When i dissolved 10 ul RIPA or 5 ul RIPA is bradford solution(from Biorad) is gives colour, though it does not havemy protein protein sample from mice kidney in that.
I want to accurately estimate the concentration of the protein in my samples and then to procced with proteomic analysis of these samples for western blot and 2-D gel.
May you could kindly guide me what should I do?
Thank you
best regards, akhtar hussain
I have extracted protein the kidney of mice with RIPA Buffer. Now I want to do the protein concentration estimation of these samples. But my sample is dissolced in RIPA buffer and this does not work accurate with bradford assay as When i dissolved 10 ul RIPA or 5 ul RIPA is bradford solution(from Biorad) is gives colour, though it does not havemy protein protein sample from mice kidney in that.
I want to accurately estimate the concentration of the protein in my samples and then to procced with proteomic analysis of these samples for western blot and 2-D gel.
May you could kindly guide me what should I do?
Thank you
best regards, akhtar hussain
Irit Rappley
Mar 7, 2013, 1:18:56 PM3/7/13
to akhatr hussain, met...@magpie.bio.indiana.edu
Hi Akhtar,
The Bradford assay can only be used if your samples have very low concentration of detergents (here is a table<http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/proteomics-and-protein/migrationpep1/compatibility-chart.Par.0001.Image.-1.-1.1.gif> showing how much detergent can be used). You should use the BCA assay instead:
http://www.piercenet.com/browse.cfm?fldID=02020101 (this is the kit that I use)
http://www.sigmaaldrich.com/catalog/product/sigma/bca1?lang=en®ion=US
Cheers,
Irit
best regards, akhtar hussain _______________________________________________
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Met...@net.bio.net<mailto:Met...@net.bio.net>
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The Bradford assay can only be used if your samples have very low concentration of detergents (here is a table<http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/proteomics-and-protein/migrationpep1/compatibility-chart.Par.0001.Image.-1.-1.1.gif> showing how much detergent can be used). You should use the BCA assay instead:
http://www.piercenet.com/browse.cfm?fldID=02020101 (this is the kit that I use)
http://www.sigmaaldrich.com/catalog/product/sigma/bca1?lang=en®ion=US
Cheers,
Irit
Methods mailing list
Met...@net.bio.net<mailto:Met...@net.bio.net>
http://www.bio.net/biomail/listinfo/methods
Wenduo Ye
Mar 11, 2013, 8:47:02 PM3/11/13
to met...@magpie.bio.indiana.edu
Did anyone here used plasmid or cDNA clones from OriGene?
is their product reliable?
Moreover, in many of their plasmid they denote the plasmid are not replicable by regular DNA purification method, is this possible? Looks to me they are just trying to get customers to buy the plasmid from them instead of purifying the plasmid indefinitely in their own bench
I attached their 'explanation below'
;Due to the inherent nature of this plasmid, standard methods to replicate additional amounts of DNA in E. coli are highly likely to result in mutations and/or rearrangements. Therefore, OriGene does not guarantee the capability to replicate this plasmid DNA. Additional amounts of DNA can be purchased from OriGene with batch-specific, full-sequence verification at a reduced cost. Please contact our customer care team at custs...@origene.com or by calling 301.340.3188 option 3 for pricing and delivery.;
>
is their product reliable?
Moreover, in many of their plasmid they denote the plasmid are not replicable by regular DNA purification method, is this possible? Looks to me they are just trying to get customers to buy the plasmid from them instead of purifying the plasmid indefinitely in their own bench
I attached their 'explanation below'
;Due to the inherent nature of this plasmid, standard methods to replicate additional amounts of DNA in E. coli are highly likely to result in mutations and/or rearrangements. Therefore, OriGene does not guarantee the capability to replicate this plasmid DNA. Additional amounts of DNA can be purchased from OriGene with batch-specific, full-sequence verification at a reduced cost. Please contact our customer care team at custs...@origene.com or by calling 301.340.3188 option 3 for pricing and delivery.;
>
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