Hello Vaheh, I tried SDS incubation/heating to quickly check whether my protein of interest is bound to the beads. This was just a preliminary approach so that I could avoid eluting from the beads at this stage. However, since I observed a strange pattern, I repeated the same test with fresh beads.
It’s a reducing SDS-PAGE. Given the high-intensity bands at 55 kDa and 25 kDa, it’s very unlikely that these are anything other than the antibody chains. This is not an artifact, as I have repeated the experiment several times and obtained the same band pattern (relative to the ladder). Best regards, Preeti On Wed, Aug 13, 2025 at 10:25 PM Oganesyan, Vaheh < [email protected]> wrote: > 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 <[email protected]> *On Behalf Of *Preeti > *Sent:* Wednesday, August 13, 2025 12:43 PM > *To:* [email protected] > *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. > > Best regards, > > Preeti > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ------------------------------ > > *Confidentiality Notice: *This message is private and may contain > confidential and proprietary information. If you have received this message > in error, please notify us and remove it from your system and note that you > must not copy, distribute or take any action in reliance on it. Any > unauthorized use or disclosure of the contents of this message is not > permitted and may be unlawful. > > ######################################################################## 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/
