Manual / Product Insert

User Guide: Melon Gel IgG Purification Kit

Version: Jan. 2015
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Manual / Product Insert

PowerEase® 300W Power Supply

Version: MAN0008988 C.00 (6 May 14)

Manual / Product Insert

Pro-Q Diamond Phosphoprotein Gel Stain

Version: 05-18-2010
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Citations & References

SYPRO orange and SYPRO red protein gel stains: one-step fluorescent staining of denaturing gels for detection of nanogram levels of protein.

  • Authors: Steinberg TH, Jones LJ, Haugland RP, Singer VL
  • Journal: Anal Biochem (1996) 239:223-237
  • PubMed ID: 8811914

Product FAQ

When running two protein gels, do I need to double the voltage?

Answer

If you are running the gels at constant voltage, you do not need to increase the voltage regardless of the number of gels. However, the resulting current and wattage observed will multiply linearly with the number of gels. Keep in mind that the expected total current for your gels should not exceed the current limit of the power supply, or else the current will plateau and the run will slow down. (For example: Recommended constant voltage for running a NuPAGE Bis-Tris gel with MES Buffer is 200 V, with a starting current of 110-125 mA/gel and end current of 70-80 mA/gel. If the power supply has a current limit of 500 mA, the maximum number of NuPAGE Bis-Tris gels that can be run at one time with full power is 500 mA/125 mA = 4 gels. Any additional gels will decrease the current per gel and increase the run time.

Answer Id: E10504

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Product FAQ

Can I use the HiMark Prestained Protein Standard with NuPAGE Bis-Tris gels and Tris-Glycine gels?

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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Manual / Product Insert

Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit

Version: P30637 02-22-2007
Catalog #

Citations & References

Applications of SYPRO orange and SYPRO red protein gel stains.

  • Authors: Steinberg TH, Haugland RP, Singer VL
  • Journal: Anal Biochem (1996) 239:238-245
  • PubMed ID: 8811917

Product FAQ

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

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 a highly mobile "leading" chloride ion present in the gel buffer and the slower "trailing" glycine ion supplied by the running buffer. These concentrated, thin bands of protein undergo sieving once they reach the resolving gel, which separates them by size.

The resolution of smaller proteins (under 5 kDa) is hindered by the continuous accumulation of free dodecyl-sulfate (DS) ions (from the SDS sample and running buffers) in the stack. 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 will also interfere with the fixing and staining process later. To solve this problem, Schagger and von Jagow replaced the trailing glycine ion with a faster moving Tricine trailing ion. Many small proteins which run with the stacked DS in the Tris Glycine system will separate from DS in the Tricine gel system, resulting in sharper, cleaner bands and better resolution.

Answer Id: E3839

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Manual / Product Insert

SYPRO Tangerine Protein Gel Stain

Version: 01-13-2001

Product FAQ

What conditions should I use to transfer my gel when using the NuPAGE Transfer buffer?

Answer

We recommend the following transfer conditions: 30 V constant for 1 hr with the expected current of 220 mA/gel (start of run) to 180 mA/gel (end of run). For an overnight transfer, we recommend using 1-15 V constant. Please refer to the Nupage Large Protein Analysis System manual for more information (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/largeproteinanalysis_man.pdf).

Answer Id: E16610

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Product FAQ

What products do you offer for 2D gel electrophoresis of proteins?

Answer

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

Answer Id: E10622

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Product FAQ

I ran my protein under native conditions on a Tris-Glycine gel. It has a pI that is higher than the pH of the Tris-Glycine transfer buffer. Can you offer some tips for transferring it?

Answer

- Increase the pH of Tris-Glycine transfer buffer to 9.2, allowing all the proteins below pI 9.2 to transfer towards the anode electrode.
- Use the Tris-Glycine transfer buffer and place a membrane on both sides of the gel. If there are any proteins that are more basic than the pH of the transfer buffer, they will be captured on the extra membrane placed on the cathode side of the gel. Both membranes can then be developed in the same manner.
- Prior to blotting, incubate the gel for 15 minutes in Tris-Glycine transfer buffer containing 0.1% SDS. The small amount of SDS will give the proteins enough charge to move unidirectionally towards the anode and in most cases, should not denature the protein. Proceed with the transfer using regular Tris-Glycine transfer buffer.

Answer Id: E11607

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Product FAQ

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?

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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