What may cause streaking on the 2nd dimension gel after IEF?
There are several reasons why streaking may occur.
(1) Sample is not completely solubilized prior to application.
(2) Sample is poorly soluble in rehydration solution.
(3) Non-protein impurities in the sample can interfere with IEF, causing horizontal streaking in the final 2-D result, particularly toward the acidic side of the gel.
(4) Ionic impurities are present in sample.
(5) Ionic detergent is present in sample.
(6) Sample load is too high.
(7) Underfocusing. Focusing time was not long enough to achieve steady state focusing.
(8) Overfocusing. Extended focusing times (over 100,000 Vh) may result in electroendosmotic water and protein movement, which can produce horizontal smearing.
What should be done?
(1) Be sure that the sample is completely and stably solubilized. Note: Repeated precipitation-resolubilization cycles produce or increase horizontal streaking.
(2) Increase the concentration of the solubilizing components in the rehydration solution.
(3) Modify sample preparation to limit these contaminants or dialyze protein.
(4) Reduce salt concentration to below 10 mM by dilution or desalt the sample by dialysis. Precipitation with TCA and acetone and subsequent resuspension is another effective desalting technique that removes lipids, nucleotides and other small molecules.
Note: Specific and non-specific losses of proteins can occur with dialysis, gel chromatography, and precipitation/resuspension of samples. If the sample preparation cannot be modified, the effect of ionic impurities can be reduced by modifying the IEF protocol. Limit the voltage to 100-150 V for 2 hours, then resume a normal voltage step program. This pre-step allows the ions in the sample to move to the ends of the IPG strip.
(5) If the ionic detergent SDS is used in sample preparation, the final concentration must not exceed 0.25% after dilution into the rehydration solution. Additionally, the concentration of the non-ionic detergent present must be at least 8 times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the proteins.
(6) Extend focusing time. Load less sample.
(7) Prolong focusing time.
(8) Reduce focusing time.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
I just set up the ZOOM IPGRunner for IEF but don't see any current running through the system. What is the issue?
A possible reason is poor contact between electrodes or incomplete circuit. Make sure that you have added 600 µL deionized water to the Electrode Wicks and the gel is exposed at the anodic and cathodic ends of the cassette. Check the power supply. Be sure to set the “Load Check” to off to enable the power supply to operate at low current.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
What are the components of the ZOOM IPGRunner Mini-Cell?
Here are the components of the ZOOM IPGRunner Mini-Cell:
ZOOM IPGRunner Core
ZOOM IPGRunner Lid
Gel Tension Wedge
Buffer Dam
Mini-Cell Chamber
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
I used the ZOOM IPGRunner system and some of my protein spots are missing or appear as a smear. Can you please offer some suggestions?
Here are the possible causes and solutions:
*Protein degradation: Add protease inhibitors during sample preparation (see manual for details).
*Different subunits: Use denaturing conditions (8 M urea).
*Incomplete equilibration: Perform equilibration as described in the manual. Increase the equilibration time.
*Protein precipitates: Increase solubilization reagents in the rehydration buffer (see manual for details. Use appropriate strips based on the pI of the protein sample.
*Low protein load: Increase the protein load. You can load up to 400 µg of fractionated protein sample per ZOOM Strip. Use an accurate and sensitive protein estimation method.
*Insensitive detection method: Use sensitive detection methods such as silver staining or immunoblotting.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
I used the ZOOM IPGRunner system and got vertical streaking. Can you please offer some suggestions?
Here are the possible causes and solutions:
*Protein has got oxidized: Include DTT in the rehydration buffer and perform the alkylation step.
*Impure solutions: Use ultrapure reagents to prepare the rehydration buffer, equilibration buffer, and buffers for SDS/PAGE.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
