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User Guide: PowerEase Touch 350W Power Supply Manual / Product Insert

  • Version: A.0
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Protocol: Protein electrophoresis and western blot recipes Product Literature

User Guide: PowerEase Touch 120W Power Supply Manual / Product Insert

  • Version: A.0
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Applications of SYPRO orange and SYPRO red protein gel stains. Citations & References

  • Authors: Steinberg TH, Haugland RP, Singer VL
  • Journal: Anal Biochem (1996) 239:238-245
  • PubMed ID: 8811917
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What is the purpose of phenol red in the sample buffers for protein electrophoresis? Product FAQ

Answer

Phenol red was originally included in the Invitrogen Novex sample buffers as a true ion-front tracking dye for the very small pore-sized gels, especially Tricine gels. Phenol red is smaller than Serva Blue G250. In high percentage gels, molecules in this size range are resolved on the basis of size, such that phenol red runs with the ion front while G250 can run as much as 2 cm behind the ion front. Conversely, in larger-pored gels, there is no size-based resolution in the size range of these dyes, and the dyes resolve on the basis of charge density, with G250 running at the ion front and phenol red migrating more slowly. Phenol red is not particularly useful in the NuPAGE system, even though it is included in the NuPAGE sample buffer for the sake of consistency, especially with the Invitrogen protein standards, which all contain phenol red.

Answer Id:: E11880

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Transfer of SDS-proteins from gel electrophoretic zones into mass spectrometry, using electroelution of the band into buffer without sectioning of the gel. Citations & References

  • Authors: Yefimov S, Yergey AL, Chrambac A
  • Journal: J Biochem Biophys Methods (2000) 42:65-78
  • PubMed ID: 10647815
Catalog # C2284

What is your recommendation for the extraction buffer for my protein sample after IEF using ZOOM IPG strips? Product FAQ

Answer

*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.

Answer Id:: E10684

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Are the NuPAGE MOPS and MES SDS running buffers compatible with N-terminal sequencing of proteins via Edman degradation? Product FAQ

Answer

The NuPAGE MOPS and MES SDS running buffers are compatible with Edman protein sequencing. Proteins can be sequenced directly from NuPAGE gels.

Answer Id:: E10544

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I stained my Invitrogen NuPAGE Bis-Tris gel with Pro-Q Emerald Glycoprotein Gel Stain and observe a very high background. What is causing this? Product FAQ

Answer

Pro-Q Emerald Glycoprotein Gel Stain may show high background staining in Invitrogen NuPAGE Bis-Tris and Tris-Acetate gels, especially in combination with MES running buffer or in gels that are nearing their expiration date. The gel background increases with acrylamide density and gradient gels will show a gradual increase in background from the top to the bottom of the gel corresponding to the acrylamide gradient. Increasing the number of washes or modifying incubation times will not help to reduce this background. Better results will be obtained with Tris-Glycine or Tricine gels. If you wish to continue using Pro-Q Emerald stain with Invitrogen NuPAGE Bis-Tris gels, we recommend using recently purchased gels and MOPS running buffer. Glycoproteins will still be detected in gels with high background, but with reduced sensitivity.

Answer Id:: E11302

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Do I need to increase the voltage when I run a 1.5 mm protein gel versus a 1.0 mm gel? Product FAQ

Answer

If you are running the gel at constant voltage, you do not need to increase the voltage regardless of the thickness of the gel.

Answer Id:: E13205

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Do you recommend adding the NuPAGE Antioxidant to the NuPAGE transfer buffer when I transfer proteins from NuPAGE Bis-Tris or NuPAGE Tris-Acetate gels? Product FAQ

Answer

Yes, we recommend adding the NuPAGE Antioxidant to the NuPAGE transfer buffer for enhanced blotting results with reduced proteins in order to maintain the reduced state of the proteins throughout the run.

Answer Id:: E10550

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I am observing large fuzzy background spots that are obscuring my protein spots after staining my 2D gels with SYPRO Ruby Protein Gel Stain. They always occur in the same position on my 2D gels. What is causing them and how can they be avoided or removed? Product FAQ

Answer

These types of spots are caused by some component of the IEF sample buffer that runs into the gel during the second dimension separation and is stained by SYPRO Ruby Protein Gel Stain. SYPRO Ruby dye is attracted to amines, such as amines in lysine, arginine and histidine containing peptides, but also amines in detergents and ampholytes. The simplest solution is to try a different IEF buffer formulation that does not cause this artifact. Possibly, a thorough overnight fixation in several changes of 50% methanol/7% acetic acid will wash out the contaminant.

Answer Id:: E11277

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Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels. Citations & References

  • Authors: Daban JR, Bartolomé S, Samsó M
  • Journal: Anal Biochem (1991) 199:169-174
  • PubMed ID: 1725949
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Can I use the HiMark Prestained Protein Standard with NuPAGE Bis-Tris gels and Tris-Glycine gels? Product FAQ

Answer

The HiMark Prestained Protein Standard can be used with NuPAGE Invitrogen 4-12% Bis-Tris Gels (with NuPAGE MOPS SDS Running Buffer) and Invitrogen 4% Tris-Glycine Gel (with Tris-Glycine SDS Running buffer). However, to obtain the best results with high molecular weight proteins, we recommend using the HiMark Prestained Protein Standard with NuPAGE Invitrogen Tris-Acetate Gels (with Tris-Acetate SDS buffer system).

Answer Id:: E11669

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