Heat may hydrolyze a protein, usually forming a smear or ladder.
0.01% SDS appears very low. Try 0.5% and see if the band patterns change. Some IgG used for making the resin may have different glycosylation. Protein may form aggregates on non-reducing gel. For reducing gel, add lots of reducing agent. --Chun From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Oganesyan, Vaheh Sent: Wednesday, August 13, 2025 1:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Unusual SDS-PAGE pattern from IgG beads Tom, You’re correct. I do (well, did) boil samples in SDS before running SDS PAGE. I take those words/sentences back. Thank you for correcting me. Vaheh From: Tom Peat <t.p...@unsw.edu.au <mailto:t.p...@unsw.edu.au> > Sent: Wednesday, August 13, 2025 4:55 PM To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> ; Oganesyan, Vaheh <vaheh.oganes...@astrazeneca.com <mailto:vaheh.oganes...@astrazeneca.com> > Subject: Re: [ccp4bb] Unusual SDS-PAGE pattern from IgG beads Hello Vaheh, Simply heating proteins shouldn't 'digest' them into different pieces. SDS PAGE gel sample preparation typically has a heating step and the whole point of the gel is to denature the protein (not digest it) so you can look at what is in the sample based roughly on size. Not all proteins behave well when run on a gel, but one shouldn't be getting digestion of proteins unless one has added protease at some point. Best regards, Tom _____ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> > on behalf of Oganesyan, Vaheh <vaheh.oganes...@astrazeneca.com <mailto:vaheh.oganes...@astrazeneca.com> > Sent: Thursday, August 14, 2025 2:55 AM To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> > Subject: Re: [ccp4bb] Unusual SDS-PAGE pattern from IgG beads Hi Preeti, 1. No IgG is meant to withstand heating to 95 degrees Centigrade, let alone in the presence of SDS. 2. It is not that easy to determine the molecular weight of proteins just by looking at SDS page, not to mention that the appearance of your bands will depend also on the presence or absence of reducing agent. 3. By heating an IgG to 95 degrees you will inadvertently digest it to many pieces. Sizes of those pieces will be different from one Ab to another. Can you explain the purpose of your protocol that includes destruction of IgG Sepharose? Vaheh From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> > On Behalf Of Preeti Sent: Wednesday, August 13, 2025 12:43 PM To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Unusual SDS-PAGE pattern from IgG beads Hi everyone, I have an off-topic doubt. I’m using Sepharose IgG beads to purify a protein with a Protein A tag. However, when I load fresh beads (without any protein) after incubating them with 0.01% SDS or heating them at 95 °C in the presence of SDS, I observe a strange band pattern on the SDS-PAGE * With only 0.01% SDS: I see two consecutive bands between 72 and 95 kDa of same intensity * With heating: I see the same two bands (same intensity as above), plus two strong bands at ~55 kDa and ~25 kDa . The 55 kDa and 25 kDa bands are clearly the heavy and light chains, but I can’t figure out what the upper bands are. I was wondering if anyone could help me understand this. 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