What does it mean when bands appear to be getting narrower (or "funneling") as they progress down a protein gel?
There may be too much beta-mercaptoethanol (BME), sample buffer salts, or dithiothreitol (DTT) in your samples. If the proteins are over-reduced, they can be negatively charged and actually repel each other across the lanes causing the bands to get narrower as they progress down the gel.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
What is meant by the terms "Straightness" and "Curvature" on the Certificate of Analysis for a Invitrogen protein gel?
Gel straightness is defined as the straightness across all lanes of the gel, measured at the bottom, expressed relative to the total length of the gel. For example, a gel with straightness of 0.020 Rf is flat to within 2% of the length of the gel (1.6 mm) across. Band curvature is defined as the curvature of the bands in the outer lanes of the gel, expressed relative to the total length of the gel. For example, bands with curvature of 0.010 Rf are straight to within 1% of the length of the gel (0.8 mm).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
What procedures are carried out for QC of Invitrogen gels?
The QC of our gels includes several processes:
1) Each gel is checked by eye for visible anomalies.
2) Under defined conditions, gels retained from each lot are tested as follows:
--When gels are run at a defined voltage, the resulting current and power of the electrophoresis are measured.
--Protein samples are electrophoresed on test gels to determine the gel run time and the protein band quality after electrophoresis. Bands are examined for: straightness within bands, curvature of bands across the gel ("smiling" or "frowning"), and reproducibility of the Rf values for protein molecular weight markers. According to these results, a Certificate of Analysis is created, which is available upon request.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
After western detection, my membrane has a lot of spots. What could have gone wrong?
Here are possible causes and solutions:
- Membrane blotting pads are dirty or contaminated. Soak pads with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
- Blocking was uneven. The incubation dish must be sufficiently big to allow thorough coverage of membrane. Shake or agitate during each step.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
I am getting a lot of non-specific binding after western detection. Can you offer some tips?
Here are possible causes and solutions:
- Membrane contaminated by fingerprints or keratin proteins: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Concentrated secondary antibody used: Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
- Concentrated Primary antibody used: Decrease the concentration of the primary antibody.
- Affinity of the primary antibody for the protein standards: Check with the protein standard manufacturer for homologies with primary antibody.
- Insufficient removal of SDS or weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection.
- Short blocking time or long washing time: Make sure that each step is performed for the specified amount of time.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
