I am trying to do a Western Blot to detect acidic proteins (~30kDa) that have a PI of ~3.5, due to the presence of Asp and Glu repeats. The proteins are about 80% pure. After I ran them on SDS-PAGE (1 µg/well), I did a wet-transfer using Tris/Glycine transfer buffer (final concentration of a 1x solution is 25mM Tris, 192 mM Glycine, 20% (v/v) methanol, 0.037% (w/v) SDS, pH 8.3, BIO-RAD) at 100mA, overnight at 4ºC. I have tried both nitrocellulose and PVDF membranes. In the case of PVDF membrane I also did the transfer without SDS in the buffer. After the transfer I could not detect the proteins in the membranes with Ponceau staining. Also, I do not detect any protein in the gel (Coomassie blue staining) after the transfer. Both protein marker and GST-fusion proteins transfer to the membranes and stain with Ponceau. Any suggestions?
I'd repeat the experiment and stain the gel before transfer to check that the proteins were separated. You could use reversible staining with Zn/imidazole so that the gel is available for transfer later (doi:10.1016/j.ab.2004.02.017)
High intrinsic negative charge could interfere with SDS binding. Then it might be advisable to perform the electrophoresis well below the pK3 of the carboxy-group (around 3). At such low pH, SDS is not very soluble, but LiDS is (Article Dodecyl sulfate/polyacrylamide gel electrophoresis at low pH...
).
you might also find this paper interesting depending on the structure of your protein
Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2
Lucía García‐Ortega Vivian De los Ríos Antonio Martínez‐Ruiz Mercedes Oñaderra Javier Lacadena Álvaro Martínez del Pozo Dr. José G. GavilanesFirst published: 16 September 2005 https://doi.org/10.1002/elps.200500261Citations: 31📷PDFTOOLS SHARE
Abstract
Ribonuclease U2 is a low‐molecular‐weight acidic protein with three disulfide bridges. This protein displays an anomalous electrophoretic behavior on standard SDS‐PAGE. The electrophoretic mobility of the nonreduced protein roughly corresponds to its molecular mass while the migration of the reduced protein would be in accordance with the expected molecular mass of the protein dimer. This study reveals that the protein does not bind SDS under the SDS‐PAGE conditions, its electrophoretic mobility being only determined by its electrostatic charge and hydrodynamic properties. In addition, the nonreduced protein cannot be blotted to a membrane. Unfolding of the protein upon reduction of its disulfide bridges enables electrotransference to membranes due to a restricted diffusion along the electrophoresis gel
Paul Rutland, firstly, thank you for the paper. In it, the authors weren't able to do the transfer because their protein had disulfide bonds, which my protein doesn't. But thank you for the insight.
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