Hi Anamika,
I have the protocol I used to do all the time when I was a wetlab
scientist in another lifetime:
https://tuilab.wordpress.com/2018/09/27/blue-native-gels/
I used to run native gels and western blot too. I used BSA as a suitable
weight marker, after transfer simply mark the BSA ladder with a ball
pen. All details in the blogpost above 😇
Best of luck!
Kind regards,
Deborah
On 01/06/2026 02:37, Anamika Singh wrote:
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.
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--
-------------------------------------------------------------------
Deborah Harrus, Ph.D.
PDBe Archive Project Leader, Biocuration Lead
Protein Data Bank in Europe
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK
http://www.PDBe.org
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