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What is the percentage of acrylamide and ampholytes in the IEF gels? Product FAQ

Answer

IEF gels contain 5% acrylamide, 2.6% bisacrylamide, and 2% ampholytes.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E3555

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What is the percent of acrylamide, cross-linker and ampholytes in your IEF gels? Product FAQ

Answer

Our IEF gels contain 5% acrylamide, 2.6% crosslinker, and 2% ampholytes.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E10630

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What is the formulation of Bolt Bis-Tris Plus gels? Product FAQ

Answer

Bolt Bis-Tris Plus gels have a proprietary acrylamide/bisacrylamide formulation that is based on the NuPAGE Bis-Tris gel chemistry but different from it.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E10517

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Do your NativePAGE gels contain a stacking gel? Product FAQ

Answer

Our NativePAGE gels do have a stacking gel. The stacking gel for these gels is a ~1 cm region at the top of the gel where the acrylamide percentage is low (4%) and constant. Below the stacking gel, the acrylamide percentage begins to increase in the gradient portion of the gel. However, the gel buffer is the same throughout the gel. So the stacking gel in NativePAGE gels is not the same as in the Laemmli system where the stacking gel has a different pH causing a decreased ion mobility for the trailing ions. Also the entire NativePAGE gel is cast in one continuous delivery due to which no demarcation line is seen between the resolving (or gradient) portion of the gel and the stacking gel.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E10743

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My protein bands in the outer lanes of the gel show a "smiling" effect. Can you please help me troubleshoot? Product FAQ

Answer

"Smiling" bands may be the result of the acrylamide in the gel breaking down, leaving less of a matrix for the proteins to migrate. We recommend checking to ensure that the gels have not been used past their expiration date.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E10578

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I stained Tris-Glycine gels with Colloidal Blue stain and noticed that the background was higher in low acrylamide percentage Tris-Glycine gels compared to high acrylamide percentage Tris-Glycine gels. Why is this and how can I resolve it? Product FAQ

Answer

Background is generally higher in gels with less than 10% acrylamide percentage due to penetration and trapping of colloids within the large pores of these gels. Excess background may be reduced by incubating the gel in 25% methanol solution until a clear background is obtained. Be aware that the dye will also be partially removed from the bands and that prolonged incubation in >25% methanol will result in complete destaining of protein bands and background.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E10613

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Do protein standards run differently on a Zymogram gel compared to a regular Tris-Glycine gel? Product FAQ

Answer

Zymogram gels are essentially Tris-Glycine gels containing the substrate. Protein standards run based solely on the percentage of acrylamide and hence should run the same in both kinds of gels. It is quite possible though that if the standard is pre-stained, the proteins will appear a different color because of the staining (or pre-staining) of the Zymogram gels.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E10737

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I see dumbbell or barbell shaped bands after protein electrophoresis. What could be causing this? Product FAQ

Answer

Barbell shaped bands are a result of loading too large of a sample volume. When a large sample volume is loaded, part of the sample tends to diffuse to the sides of the wells. When the run begins and the sample moves through the stacking portion of the gel, the sample will incompletely stack causing a slight retardation of the portion of the sample that diffused to the sides of the wells. This effect may be intensified for larger proteins, whose migration is more impeded in the low concentration acrylamide of the stacking gel. To alleviate the problem, we recommend concentrating the protein and loading a smaller volume. This gives a "thinner" starting zone.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E10577

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What percentage of stacking gel is in Invitrogen Tris-Glycine and NuPAGE Invitrogen gels? What is the length of stacking gel? Product FAQ

Answer

The stacking gel acrylamide concentration for Tris-Glycine and NuPAGE Bis-Tris gels is 4%. For NuPAGE Tris-Acetate gels it is 3.2%.

The stacking portion is approximately 8 to 9 mm long (it ends right above the first ridge on the cassette) on most Invitrogen protein gels (i.e., Tris-Glycine and NuPAGE gels).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E3838

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What is the liquid packaged with the Invitrogen gels? Product FAQ

Answer

Invitrogen gels are packaged in Packaging Buffer: Tris HCl, pH 8.65, with 0.02% sodium azide (expect that residual acrylamide monomer is also present). Wear gloves at all times when handling gels.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E3595

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Why is the pI performance range for Invitrogen pH 3-10 IEF gels from pI 3.5-8.5 and not from pI 3-10 as the name suggests? Product FAQ

Answer

The nomenclature of our IEF gels refers to the pI range of ampholytes that are in the IEF gels, and not necessarily the focusing performance range. In practice, a pH 3-10 IEF gel can only resolve proteins with a pI up to about 8.5. This is due to "cathodic drift". During electrophoresis, acrylamide hydrolyzes to polyacrylic acid. This happens to a greater extent at higher pHs than at lower pHs. Consequently, the lower pH acid groups formed at the high pH end of the gradient titrate out the basic groups, lowering the pH gradient at the basic end of the gel to about 8.5. Basic proteins with a pI of 8.5 to 10.0 may migrate into the gel initially, but will migrate backwards out of the gel as the run proceeds.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E10644

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What causes dumbbell- or barbell-shaped bands during protein electrophoresis? Product FAQ

Answer

Barbell-shaped bands are a result of loading too large a sample volume.

When a large sample volume is loaded, part of the sample tends to diffuse to the sides of the wells. When the run begins and the sample moves through the stacking portion of the gel, the sample will stack incompletely, causing a slight retardation of the portion of the sample that diffused to the sides of the wells.

This effect may be intensified in larger proteins, whose migration is more impeded in the low concentration acrylamide of the stacking gel.

To alleviate the problem, concentrate the protein and load a smaller volume. This gives a "thinner" starting zone.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E3832

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Why is a protein with a pI greater than 8.5 unable to be focused in a vertical IEF gel, pH 3-10? Product FAQ

Answer

The nomenclature of the vertical IEF gels refers to the pI range of ampholytes that are added to the IEF gels, not necessarily the focusing performance range. In practice, a 3-10 IEF gel can only resolve proteins with a pI of up to about 8.5. This is due to "cathodic drift". During electrophoresis, acrylamide hydrolyzes to polyacrylic acid. This happens to a greater extent at higher pH than at lower pH. Consequently, the lower pH acid groups formed at the high pH end of the gradient titrate out the basic groups, lowering the pH gradient at the basic end of the gel to about 8.5. Basic proteins with a pI of 8.5 to 10.0 may migrate into the gel initially, but will migrate backwards out of the gel as the run proceeds, and even if the proteins were focused at the pIs outside of the performance range, they will not hold.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E3553

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What is Thermo Scientific Tetramethylethylenediamine (TEMED)? Product FAQ

Answer

Thermo Scientific Tetramethylethylenediamine (TEMED) is an essential catalyst for polyacrylamide gel polymerization. TEMED is used with ammonium persulfate (APS) to catalyze acrylamide polymerization when preparing gels for electrophoresis.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E13366

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I performed a semi-dry transfer using the Invitrogen Semi-Dry Blotter and there was insufficient binding of proteins to the membrane. Can you offer some tips? Product FAQ

Answer

Here are possible causes and solutions:

- Air spaces are interfering with contact between the gel and the membrane: Roll the membrane with a blotting roller (or a clean test tube or pipet) before placing the gel on the membrane, and then remove any air bubbles between the gel and membrane with a blotting roller or a wet gloved finger. Transfer will not occur where the gel is not in contact with the membrane.
- Electrophoretic conditions were incorrect or not ideal: Running conditions, sample preparation, percentage acrylamide, and many other variables can affect the migration and resolution of proteins. Please review your electrophoresis conditions.
- Under- or over-compression of gel: Follow the compression guidelines in the manual (http://tools.thermofisher.com/content/sfs/manuals/Invitrogen_semidry_blotter_man.pdf). Too little compression can allow proteins to migrate between the gel and membrane, causing protein band smearing. Too much compression can distort the gel.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E11610

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