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User Guide: Unstained Protein Molecular Weight Marker Manual / Product Insert

  • Version: B.0
Catalog # 26610

User Guide: Pierce Prestained Protein Molecular Weight Marker Manual / Product Insert

  • Version: C.0
Catalog #

Protein Molecular Weight Standards Manual / Product Insert

  • Version: 01-13-2001
Catalog # P6649

How should the stock solution of Protein Molecular Weight Standards (broad range) be stored? Product FAQ

Answer

For long-term storage, store the stock tube at either -20 degrees C or -80 degrees C. For short-term storage, store at 2-6 degrees C. The stock can be subjected to repeated freezing and thawing without any deleterious effects to the molecular weights of the proteins or their migration during electrophoresis.

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

Answer Id: E11737

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What are the characteristics of the proteins included in the Protein Molecular Weight Standards (broad range)? Product FAQ

Answer

All proteins are single chain, monomeric proteins (no subunits). Ovalbumin is a serine-phosphate with 2 phosphorylated residues.

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

Answer Id: E11738

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How much protein is in the stock solution of Protein Molecular Weight Standards (broad range)? Product FAQ

Answer

Each of the eleven proteins is present at approximately 60 µg/mL concentration.

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

Answer Id: E11736

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What procedures are carried out for QC of Invitrogen gels? Product FAQ

Answer

The QC of our gels includes several processes:

1) Each gel is checked by eye for visible anomalies.

2) Under defined conditions, gels retained from each lot are tested as follows:

--When gels are run at a defined voltage, the resulting current and power of the electrophoresis are measured.

--Protein samples are electrophoresed on test gels to determine the gel run time and the protein band quality after electrophoresis. Bands are examined for: straightness within bands, curvature of bands across the gel ("smiling" or "frowning"), and reproducibility of the Rf values for protein molecular weight markers. According to these results, a Certificate of Analysis is created, which is available upon request.

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

Answer Id: E3586

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I am using the NorthernMax Kit. Why is my molecular weight marker and sample RNA stained poorly with ethidium bromide? Product FAQ

Answer

In order to avoid heat-induced strand scission of RNA that occurs in the presence of divalent cations (e.g., Mg++), we have increased the EDTA content of the Formaldehyde Load Dye supplied with the NorthernMax and NorthernMax Plus Kits to a level significantly higher than the formulations found in many common laboratory guides.

When ethidium bromide is added directly to the Formaldehyde Load Dye before electrophoresis, the ethidium staining of the RNA is reduced compared to typical dye formulations. There is no reduction in ethidium bromide staining when gels containing samples run with Formaldehyde Load Dye are stained post-electrophoresis.

Another effect of the relatively high EDTA concentration in the Load Dye is that the sensitivity of the positive control reaction supplied with the kit is increased relative to typical formaldehyde loading dyes. In other words, when the Control Template RNA from the kit is electrophoresed, blotted and probed with either RNA or DNA probes produced using the control templates from the kit, a stronger signal is seen from samples run in Ambion Formaldehyde Load Dye than those run in loading dyes with less EDTA.

Answer Id: E19233

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I am using the NorthernMax-Gly Kit. Why is my molecular weight marker and sample RNA stained poorly with ethidium bromide? Product FAQ

Answer

Although there is some ethidium bromide in the Glyoxal Load Dye, additional ethidium bromide may be required for optimal staining. We recommend post-staining samples by incubating in running buffer containing 0.5 µg/mL ethidium bromide to increase the fluorescence of the RNA.

Answer Id: E19245

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Some of my pre-stained molecular weight markers are appearing as dark bands when I stain the gel with SYPRO Ruby Protein Gel Stain. What is causing this? Product FAQ

Answer

Blue-colored dyes absorb light in the red wavelengths, so they absorb the red fluorescent emission of SYPRO Ruby dye. SYPRO Ruby dye still binds these proteins, but the signal is quenched by the colored dye, resulting in a negatively stained, dark band. Examples of molecular weight markers with blue-colored proteins that will quench SYPRO Ruby fluorescence are the BenchMark Pre-Stained Protein Ladder and some proteins in the SeeBlue Plus2 Pre-Stained Standard. The same phenomenon can be seen with the bromophenol blue dye front, if it is not completely run off the gel, and loss of signal when SYPRO Ruby stained gels are subsequently stained with Coomassie Blue stains. Most other colored dyes do not quench the SYPRO Ruby dye signal and will appear as normally stained protein bands.

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

Answer Id: E11278

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High Molecular Weight DNA Markers Manual / Product Insert

  • Version: 05/09/03
Catalog # 15618010

May I use the pHrodo Deep Red Antibody Labeling Kit (Cat. Nos. P35355, P35356) to label other proteins that are not antibodies? Product FAQ

Answer

Yes. The pHrodo Deep Red Antibody Labeling Kit (Cat. Nos. P35355, P35356) was optimized to label either 100 µg or 1 mg of a ~150 kDa protein (molecular weight of a whole IgG) but can be used to label other proteins. Optimize the mass of protein to label based on the molecular weight of the protein relative to the molecular weight of a whole IgG (~150 kDa).

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

Answer Id: E22095

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How do I confirm that my protein sample is crosslinked? Product FAQ

Answer

Crosslinking on a protein of interest is commonly confirmed via SDS-PAGE. The crosslinked protein molecular weight should be larger as indicated by reduced gel mobility after crosslinking when compared to an unmodified control sample.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Answer Id: E18893

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Can I use other molecular weight standards with the Pro-Q Diamond Phosphoprotein stain? Product FAQ

Answer

Other known phosphoproteins can be used as positive control standards for the Pro-Q Diamond Phosphoprotein stain. Ovalbumin, in the Protein Molecular Weight Standards Reagent (Cat. No. P6649) is a phosphoprotein. None of the proteins in the Mark12, Invitrogen Sharp, SeeBlue or SeeBlue Plus2 standards is a phosphoprotein that could be used as a positive control with the Pro-Q Diamond Phosphoprotein stain.

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

Answer Id: E11199

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How can I improve the signal intensity when using WesternDot detection reagents? Product FAQ

Answer

Here are possible causes and solutions for weak/no signal:

- Poor or incomplete transfer: Check transfer conditions, and repeat blot. Use positive control and/or molecular weight marker.
- Nitrocellulose membrane not completely wetted, or PVDF membrane not completely reactivated: Follow instructions for pre-wetting or reactivating the membrane.
- Secondary antibody concentration too low: Use the recommended secondary antibody concentrations.
- Primary antibody concentration too low: Use twice the concentration of primary antibody required for a standard immunodetection. If the signal is still low and the background is not high, increase the concentration.
- Inactive primary antibody: Determine activity by performing a dot-blot or other methods.
- Low affinity of primary antibody to antige: Obtain a higher affinity primary antibody.
- Sample improperly prepared; antigenicity weakened, or destroyed: SDS and reducing agents may interfere with some antibody/antigen affinities.
- Sample too dilute: Load a higher concentration or amount of protein onto the gel.
- Blots are too old: Protein may have broken down over time. Use freshly prepared blots.
- Protein of interest ran off the gel: Match gel separation range to the size of the protein being transferred.
- Poor retention of proteins: Larger proteins require more transfer time, while smaller proteins require less transfer time. Use a molecular weight marker with relevant size proteins. Use membrane with the appropriate binding capacity.
- WesternDot reagents have been frozen: Qdot probes will irreversibly aggregate at freezing temperatures

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

Answer Id: E11328

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