Ive always quenched and loaded directly. Never had a problem (for methylation reactions that are done in aldehydes) . However, you should ensure the column is already cleaned before and I would advise you to clean it after as well with NaOH, just in case you have excess glutaraldehyde and proteins bound on column.
-- Kelvin Lau Protein production and structure core facility - PTPSP EPFL SV PTECH PTPSP AI 2146 (Bâtiment AI) Station 19 CH-1015 Lausanne Switzerland Email: [email protected]<mailto:[email protected]> Phone: +41 21 69 34494 On 21 Jan 2022, at 15:59, Maria Jose Sanchez Barrena <[email protected]<mailto:[email protected]>> wrote: Dear all: Can I ask for some help-opinion on possible problems for a superdex200 column to separate a glutaraldehyde-crosslinked sample? We are using 0.1% Glutaraldehyde to crosslink and protein complex and after incubation, we stop the reaction by adding 50mM Tris buffer. We have analyzed the sample on an SDS-PAGE gel and we do not observe aggregates, only the crosslinked complex and some uncrosslinked protein. We would like to further analyze this sample on a gel filtration Superdex 200 Increase 3.2/300 column and although I cannot see any incompatibility in the column specifications, I would like to have some advice on how you deal with this. Since the glutaraldehyde is quenched with Tris, I imagine that passing this sample through the column would not make any harm in future purifications or will have a bad effect on resin stability. Would I need to wash the column with something special? Do you have columns dedicated to glutaraldhyde-containing samples? I am a bit concerned on “contaminating” the column or losing resolution in the future. Many thanks in advance and all best wishes for 2022, María J. Sánchez-Barrena ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
