Hi All, I am currently working with fibroblast cells for my experiments, which results in a limited amount of protein after lysis. The protein of interest is more than 150 kDa. Below is the protocol I am using:
1. After collecting the samples at various time points, I lyse them using RIPA Buffer containing 1 mM EDTA, 1X Protease Inhibitor (from Thermo), Sodium Orthovanadate, and Sodium Fluoride. 2. I perform a BCA assay and prepare the samples with 2X NATIVE PAGE Buffer (Bio-Rad). 3. I prepare 1X Tris/Glycine Buffer for the anode (outer chamber) and 1X Tris/Glycine Buffer with 1% sodium deoxycholate for the cathode (inner chamber). 4. I run the gels at 80 mA in a cold room overnight for a total of four gels. 5. The gel is then transferred using 1X Tris/Glycine Buffer with 20% methanol for about 45 minutes at 100 V. I am having difficulty resolving monomers and oligomers on the blot. Additionally, I would like to know if there is a suitable molecular weight marker for these experiments. So far, I have used both the unstained and dual-color precision markers from Bio-Rad, which I believe are compatible with SDS-PAGE. After transferring the gel, how can I determine whether the bands I see are oligomers or monomers without a marker? I have another question regarding the storage of NATIVE samples. As far as I know, we cannot store the samples as high-order oligomers tend to dissociate if frozen and thawed. I would appreciate any insights into possible steps I might be taking incorrectly. I am interested in hearing from anyone who has used the NativeMark™ Unstained Protein Standard from Invitrogen. I would appreciate your experiences with it. I am currently conducting Western Blotting, and I am curious about how the transfer works with this unstained standard. Looking for insightful responses. Thank you. Best, Anamika Singh, Ph.D. ######################################################################## 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/
