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Brochure: Clone. Purify. Analyze. (Singapore) Product Literature

If a protein sample was originally solubilized in SDS, can it be run on an IEF gel? Product FAQ

Answer

No, the SDS will render the protein complex a strong negative charge and therefore an unreliable separation based on isoelectric point. If you have a protein that is moderately soluble, then you can try to solubilize the protein in a non-ionic detergent (between 0.1-0.5%) such as Triton X-100, NP-40, or Tween 20. The non-ionic detergent may help to solubilize the protein, however, this needs to be tested empirically, because the characteristics of the protein such as its purity, concentration, and ionic strength will influence how well it will run on the gel. Very hydrophobic proteins, like membrane proteins, will not work well, even if a non-ionic detergent is added to the sample buffer.

Answer Id:: E10649

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Which pre-stained protein standards are compatible with Bolt Bis-Tris Plus gels? Product FAQ

Answer

SeeBlue prestained standard, SeeBlue Plus 2 prestained standard and Invitrogen Sharp prestained standard are compatible with Bolt Bis-Tris Plus gels. Their migration is the same as that in NuPAGE gels with the corresponding buffer.

Answer Id:: E10528

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What are the recommended sample loading volumes and protein loading amounts for Tricine gels? Product FAQ

Answer

The recommended sample loading volumes and protein loading amounts for the different well formats are listed on Page 8 of the Invitrogen Pre-Cast Gel Electrophoresis Guide (https://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf).

Answer Id:: E10721

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The protein bands in some of my gel lanes are irregular or wavy? What would have caused this problem? Product FAQ

Answer

This could be due to:

*Debris in the well
*High salt in the sample (make sure that the salt concentration does not exceed 50-100 mM)
*Running buffer issue
*Gel casting error

Answer Id:: E10586

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Can I run reduced and non-reduced protein samples on the same gel? Product FAQ

Answer

We do not recommend running reduced and non-reduced protein samples on the same gel, especially in adjacent lanes, since the reducing agent may have a carry-over effect on the non-reduced samples if they are in close proximity.

Answer Id:: E10502

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After protein electrophoresis on a Precise Tris-HEPES gel, the buffer front and proteins migrated only partly, and the bands were distorted. What could have gone wrong? Product FAQ

Answer

Please make sure that the Tris-HEPES SDS running buffer is used with these gels. The Precise Tris-HEPES gels are designed to be used with the Tris-HEPES SDS running buffer for optimal speed and resolution. We do not recommend using the Tris-Glycine SDS running buffer as this will result in poor migration and band resolution.

Answer Id:: E10620

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What gels can I use to separate native proteins? Product FAQ

Answer

The NativePAGE Invitrogen Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analyzing native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessing purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow.

Answer Id:: E5195

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Can I use 2-D gels to analyze proteins from cells labeled by SILAC? Product FAQ

Answer

Yes. Samples labeled by SILAC can be analyzed by 2-D or in conjunction with 1-D gels to validate results, analyze difficult proteins, and screen for specific proteins that are hard to identify by 1-D SDS-PAGE.

Answer Id:: E4883

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I ran my protein samples on a Tris-Glycine gel and the samples stopped running in the bottom portion of the gel whereas the marker ran fine. Why did this happen? Product FAQ

Answer

This can happen if Tris-Glycine gels are run using NuPAGE Running buffer containing Antioxidant. Please make sure that the correct Tris-Glycine Running buffer is used with Tris-Glycine gels.

Answer Id:: E10607

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Why do Tricine gels work better for smaller proteins and peptides? Product FAQ

Answer

The Tricine gel system, first described by Schagger and von Jagow in 1987, is a modification of the Laemmli Tris-Glycine system to allow for better resolution of smaller proteins and peptides. In the Laemmli system, the proteins are "stacked" in the porous, top portion of the gel (stacking gel) between the highly mobile "leading" chloride ions, present in the gel buffer and the slower "trailing" glycine ions, supplied by the running buffer. These stacked protein bands undergo sieving once they reach the separating gel, thus resolving by size. However, the resolution of smaller proteins (under 10 kDa) is hindered by the continuous accumulation of free dodecyl-sulfate (DS) ions (from the SDS sample and running buffers) in the stacking gel. This build-up of DS leads to convective mixing of the DS ions with the smaller proteins, causing fuzzy bands and decreased resolution. The mixing of the DS ions with the small proteins also interferes with the fixing and staining process later.

To solve this problem, we offer the Invitrogen Tricine gel system that is based on the Tris-Glycine system developed by Schagger and von Jagow. This modified system uses a low pH in the gel buffer and substitutes the trailing glycine ions with faster moving tricine trailing ions. Many small proteins and peptides that migrate with the stacked DS micelles in the Tris-Glycine system are now well separated from DS ions in the Tricine gel system, resulting in sharper, cleaner bands and higher resolution.

Answer Id:: E10717

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Which protein standard do you recommend using with gels run under native conditions? Product FAQ

Answer

We recommend using the NativeMark Unstained Protein Standard, Cat. No. LC0725.

Answer Id:: E10503

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What is 2D protein gel electrophoresis? Product FAQ

Answer

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.

Answer Id:: E10621

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What is meant by the terms "Straightness" and "Curvature" on the Certificate of Analysis for a Invitrogen protein gel? Product FAQ

Answer

Gel straightness is defined as the straightness across all lanes of the gel, measured at the bottom, expressed relative to the total length of the gel. For example, a gel with straightness of 0.020 Rf is flat to within 2% of the length of the gel (1.6 mm) across. Band curvature is defined as the curvature of the bands in the outer lanes of the gel, expressed relative to the total length of the gel. For example, bands with curvature of 0.010 Rf are straight to within 1% of the length of the gel (0.8 mm).

Answer Id:: E3587

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Do you offer staining trays for staining protein gels? Product FAQ

Answer

We offer the following staining trays for staining protein gels:
- StainEase Staining Trays (Cat. No. NI2400) for convenient and even staining of Mini and Midi gels. The staining tray assembly includes a perforated strainer insert that holds the gel and drains away the stain, a lid, and an outer container in which stains and solutions can be changed without having to handle the gel. Each tray easily fits up to 4 mini gels.
- Mini Gel Incubation Trays (Cat. No. 22843) for staining mini gels and western blot membranes.

Answer Id:: E10972

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