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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.

Answer Id:: E13366

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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.

Answer Id:: E3553

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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.

Answer Id:: E10644

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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.

Answer Id:: E11610

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Separation of amino acid homopolymers by capillary gel electrophoresis. Citations & References

  • Authors: Dolník V, Novotny MV
  • Journal: Anal Chem
  • PubMed ID: 8452245
Catalog #
  • A6222
  • A2333(Discontinued)

Dynamic changes of red cell membrane thiol groups followed by bimane fluorescent labeling. Citations & References

  • Authors: Kosower NS, Kosower EM, Zipser Y, Faltin Z, Shomrat R
  • Journal: Biochim Biophys Acta
  • PubMed ID: 7213703
Catalog #

Synthesis and properties of acrylamide-substituted base pair specific dyes for deoxyribonucleic acid template mediated synthesis of dye polymers. Citations & References

  • Authors: Bünemann H, Dattagupta N, Schuetz HJ, Müller W
  • Journal: Biochemistry
  • PubMed ID: 7248253
Catalog # M1698

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.

Answer Id:: E10577

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Application of capillary gel electrophoresis to the diagnosis of the aldehyde dehydrogenase 2 genotype. Citations & References

  • Authors: Tomita M; Okuyama T; Hidaka K; Ameno S; Ameno K; Ijiri I
  • Journal: Journal of Chromatography. B, Biomedical Applications
Catalog #

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis. Citations & References

  • Authors: Chen DY, Swerdlow HP, Harke HR, Zhang JZ, Dovichi NJ
  • Journal: J Chromatogr
  • PubMed ID: 1761625
Catalog #

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.

Answer Id:: E3832

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A comparison of intact human red blood cells and resealed and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes. Citations & References

  • Authors: Cabantchik ZI, Balshin M, Breuer W, Markus H, Rothstein A
  • Journal: Biochim Biophys Acta
  • PubMed ID: 1125247
Catalog # D337(Discontinued)

In polyacrylamide gel electrophoresis, why do my oligos not run at the expected size? Product FAQ

Answer

While not the case for large DNA molecules, the migration of oligonucleotides in polyacrylamide gels is affected by the sequence and composition. Comparing an oligo to a molecular size standard or another oligonucleotide of known size is not a reliable way to determine its size.

The addition of 40% formamide (in addition to 8 M urea) to the acrylamide gel solution removes most sequence-dependent migration effects. [Reference: Ausubel, F.M., Brent, R., Kingston, R.E. Moore, D.D., Seidman, J.G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York, NewYork.]

In addition, the use of ethidium bromide for detection and gel densitometry-based quantitation of an oligonucleotide is not reliable. The ability of ethidium bromide to stain oligonucleotides is poor and varies depending on sequence and composition.

Answer Id:: E4546

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Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Citations & References

  • Authors: McMaster GK, Carmichael GG
  • Journal: Proc Natl Acad Sci U S A
  • PubMed ID: 73185
Catalog #

Organization of the inter-alpha-inhibitor heavy chains on the chondroitin sulfate originating from Ser(10) of bikunin: posttranslational modification of IalphaI-derived bikunin. Citations & References

  • Authors: Enghild JJ, Thøgersen IB, Cheng F, Fransson LA, Roepstorff P, Rahbek-Nielsen H
  • Journal: Biochemistry
  • PubMed ID: 10512637
Catalog # A350
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