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.
What is the protocol for using ZOOM gels after first-dimension separation on an IPG strip?
1) Trim excess plastic from IPG strip
2) Equilibrate IPG strip for 2 x 15 min in 5 mL of the appropriate buffer (Use NuPAGE sample buffer for NuPAGE ZOOM gels and Tris Glycine ZOOM gels). Equilibration buffer: 1X NuPAGE sample buffer, 50 mM DTT.
3) Place equilibrated IPG strip in large well of ZOOM gel. Seal the strip in place with an agarose (0.5%) overlay.
4) Load molecular weight markers in small well of ZOOM gel.
Variations: To alkylate the proteins after reducing them, prior to separation on ZOOM gels: 1) Incubate IPG strip for 15 min in equilibration buffer 2) Transfer strip to Equilibration Buffer containing 125 mM iodoacetamide and lacking reducing agent. Incubate an additional 15 min. To denature the proteins with urea after IEF: 6 M urea can be added to Equilibration Buffer, if desired.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
How should I equilibrate ZOOM strips before performing SDS-PAGE using ZOOM gels?
Incubating ZOOM Strips in NuPAGE LDS Sample Buffer equilibrates the strips in SDS buffer and prepares the strips for 2D SDS-PAGE.
We recommend using the NuPAGE LDS Sample Buffer containing 50 mM DTT (NuPAGE Sample Reducing Agent) with NuPAGE Invitrogen 4-12% Bis-Tris ZOOM Gels and Invitrogen 4-20% Tris-Glycine ZOOM Gels. You need 5-15 mL of buffer per equilibration tray. For alkylation, we recommend incubating the strips in 125 mM Alkylating Solution (prepared by dissolving 232 mg of fresh iodoacetamide in 10 mL of 1X NuPAGE LDS Sample Buffer).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
What is your recommendation for the extraction buffer for my protein sample after IEF using ZOOM IPG strips?
*The rehydration buffer is the best extraction buffer for IPG strips.
*The ionic strength of the buffer should be low; e.g., PBS will not work because the ionic strength is pretty high (150-250 mM), depending on salt and other ionic components.
*Proteins can be precipitated and re-suspended in rehydration buffer; use acetone for precipitation.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Can I rehydrate my ZOOM IPG strip for less than 8-16 hours?
Our proprietary formulation for our ZOOM Strips has been tested and been shown to allow for a 60-90 minute rapid rehydration step. It is not necessary to go overnight, although for convenience, you still can rehydrate for 8-16 hours. For the one hour rehydration, it is important not to exceed the 140 µL sample volume. The proteins get in very quickly but the liquid may be left behind. If rehydrating overnight, use the 155 µL sample volume.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
