From snipeurope from hotmail.com Mon Aug 3 13:25:21 2009 From: snipeurope from hotmail.com (=?iso-8859-1?Q?Pepa_Florez_P=E9rez?=) Date: Mon Aug 3 15:41:01 2009 Subject: [Bioforum] Pressure limit AND Ni-NTA beads Message-ID: Hello everybody, here it is my first question. I am using Ni-NTA agarose beads to purify a protein. According to the manufacturer the limit pressure is around 2 psi (nothing), and I am running the column to 50 psi. Can anyone please tell me about why does the pressure limit is important when dealing with affinity chomatography? How can this (pressure limit too high) affect my purification? Am I destroying the beads? I am running the column (home-prepacked tricorn) on an akta-prime. I would really appreciate any information since I couldn't find any info on the web. Thank you very much, Eva _________________________________________________________________ Internet Explorer 8 m?s sencillo y seguro ?Desc?rgatelo gratis! http://events.es.msn.com/noticias/internet-explorer-8/ -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/bioforum/attachments/20090803/38d7a718/attachment.html From engelbert_buxbaum from hotmail.com Tue Aug 4 14:40:45 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Aug 4 16:26:59 2009 Subject: [Bioforum] Re: Pressure limit AND Ni-NTA beads References: Message-ID: Am 03.08.2009, 14:25 Uhr, schrieb Pepa Florez P?rez : > Can anyone please tell me about why does the pressure limit is important > when dealing with affinity chomatography? > How can this (pressure limit too high) affect my purification? > Am I destroying the beads? When you expose your beads to too high a pressure, they get compressed, and as a result packed more tightly. That reduces the flow rate of the column. You may also affect the pore size of the gel, so that fewer active groups are accessible to the protein -> reduced capacity. If I did the math correctly, 2 psi corresponds to about 0.13 bar or 1.3 m of water column, which is low even for makeshift setups with gravity feed. Probably simple agarose. I would switch to a stronger (crosslinked) gel like Sephacryl.