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 <[email protected]> on behalf of Oganesyan, Vaheh <[email protected]> Sent: Thursday, August 14, 2025 2:55 AM To: [email protected] <[email protected]> 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 <[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 Confidential communication - This email and any files transmitted with it are confidential and are intended solely for the addressee. If you are not the intended recipient, please be advised that you have received this email in error and that any use, dissemination, forwarding, printing, or copying of this email and any file attachments is strictly prohibited. If you have received this email in error, please notify me immediately by return email and destroy this email. ######################################################################## 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/
