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View additional product information for ZOOM™ IPG Strips pH 9-12 - FAQs (ZM0017)
17 product FAQs found
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.
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.
*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.
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.
We do not recommend heating protein samples containing urea over 37 degrees C as elevated temperatures cause urea to hydrolyze to isocyanate, which modifies proteins by carbamylation.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We recommend storing them at -80 degrees C. We do not recommend storing them at -20 degrees C
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
ZOOM Strips pH 9-12 offer a linear pH range of 9-12 that is ideal for isoelectric focusing (IEF) and analysis of basic proteins such as nucleic acid binding proteins, transcription factors, and ribosomal proteins
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The recommended ampholyte concentration in the sample rehydration buffer is 0.5%.
*If you are loading 5-50 µg of protein (pure protein or crude lysate) per ZOOM strip, use 0.5% ampholytes in the sample rehydration buffer.
*If you are loading >50 µg of protein (crude lysate or fractionated sample) per ZOOM Strip, use 0.5-2% ampholytes in the sample rehydration buffer.
Note: Higher ampholyte concentration requires longer focusing times.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
You can store the whole cassette at -80 degrees C in a sealed container until ready to use. However, it is possible to put the individual strips in 15 mL conical tubes and freeze them at -80 degrees C. When you are ready to equilibrate the strip before running the second dimension, you can add the equilibration buffer directly to the tube. In addition, you can remove the film cover from the cassette and store the strips, in the cassette, in a zip-sealed bag for ease of removal of single strips. Do not allow the other strips to thaw and then refreeze because it might result in diffusion of the bands. The strips can be removed frozen.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The maximum volume of the protein sample should at most be 1/6 of the final sample volume that will be added to the strip. A good general volume would be 5-10µL. 140 µL of sample diluted in Sample Rehydration buffer is used to rehydrate each ZOOM Strip for the standard rehydration time of one hour. For overnight rehydration, we recommend using 155 µL of diluted sample.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We recommend starting with 5-15 µg (for silver staining) or 20-50 µg (for Coomassie staining) of total protein per ZOOM Strip. The total protein load can be increased after optimizing the sample preparation protocol and focusing parameters. Higher amounts of sample may be loaded on narrow pH range ZOOM Strips. For the ZOOM Strip pH 9-12, 50-100 µg (for silver staining) or 100-200 µg for (Coomassie staining) of total protein per strip is recommended. If the sample is a fractionated or partially purified protein, up to 400 µg of total protein per strip may be loaded.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We recommend storing the ZOOM Strips at -20 degrees C.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Alkylation prevents unwanted protein modifications by alkylating cysteines to avoid mixed disulfide formation and reoxidation and this allows for crisper focusing.
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Sorry we have discontinued selling the ZOOM 2D Protein Solubilizers.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The main advantages of performing 2D gel electrophoresis of proteins and applications used for are listed below:
Advantages:
*Simultaneous separation of hundreds to thousands of proteins
*High capacity with superior resolution
*Compatible with further analysis by MS for protein identification and sequencing
Ability to separate and analyze low-abundance proteins
Applications:
*Comparative proteomics: identifying and analyzing differences between complex mixtures of proteins
*Protein profiling, biomarker discovery
*Separation and analysis of protein variants and isoforms
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We offer the following products for the first- and second-dimension separation of proteins:
First-dimension separation:
*Vertical gels for separation of proteins based on their isoelectric point (pI) (https://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/protein-gel-electrophoresis/protein-gels/specialized-protein-separation/isoelectric-focusing.html)
*Solution-phase isoelectric focusing of proteins using ZOOM Disks (immobilized buffer disks of specific pH) to reduce sample complexity, enrich low-abundance proteins, and increase the dynamic range of detection (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/isoelectric-focusing/zoom-ief-fractionator.html)
*Mini gel system for high-throughput isoelectric focusing of proteins using ZOOM IPG (Immobilized pH Gradient) Strips
(https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/2d-gel-electrophoresis/zoom-ipgrunner-system.html)
Second-dimension separation:
*ZOOM gels for 2D electrophoresis: NuPAGE Bis-Tris (Cat. No. NP0330BOX) and Tris-Glycine (Cat. No. EC60261BOX) mini gels with IPG wells ( to accommodate 7 cm ZOOM strips) for separation of proteins based on their molecular weight
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
2D protein gel electrophoresis is the separation of proteins in two dimensions. In the first dimension, proteins are separated by their isoelectric point (pI) using isoelectric focusing, and in the second dimension, they are separated by their mass using SDS-PAGE.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.