What staining method do you recommend for NativePAGE™ gels?

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NativePAGE™ Invitrogen™ Bis-Tris Gels are compatible with most of the standard Coomassie™ R-250 or G-250 staining formulations. The Invitrogen™ Colloidal Blue Staining Kit (Cat. No. LC6025) is recommended for the most sensitive Coomassie™ staining in NativePAGE™ Gels. SimplyBlue™ Safe Stain (Cat. No. LC6060) is not recommended for use with NativePAGE™ gels, due to special fixing requirements. Both the SilverQuest™ Silver Staining Kit (Cat. No. LC6070) and SilverXpress™ Silver Staining Kit (Cat. No. LC6100) are suitable for staining of NativePAGE™ Gels. However, for best overall results and lower background, we recommend the SilverQuest™ Kit as the best choice.

Answer Id: E10753

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Which transfer buffer do you recommend using for blotting of NativePAGE™ gels?

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We recommend using the NuPAGE™ Transfer buffer for blotting of NativePAGE™ gels. PVDF is the recommended blotting membrane and works well in terms of transfer and detection. Nitrocellulose is not compatible with blotting of NativePAGE™ gels, as the nitrocellulose membrane binds the Coomassie™ G-250 dye very tightly and is not compatible with alcohol-containing solutions needed to destain the membrane and fix the proteins.

Answer Id: E10754

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What is the TaqMan™ MicroRNA Assay Index File?

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The TaqMan™ MicroRNA Assay Index File contains a table listing available TaqMan™ MicroRNA Assays and includes Part Number (a.k.a. Catalog Number), miRNA sequence, TaqMan™ MicroRNA assay name, miRBase name and accession numbers, and a cross species chart. The file is available on the product page and can be easily downloaded from http://www.appliedbiosystems.com/support/miRNA_aif.xls. The file is updated regularly, as new assays are released and when miRBase is updated.

Answer Id: E2763

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What does it mean when bands appear to be getting narrower (or “funneling”) as they progress down a protein gel?

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There may be too much BME (beta-mercaptoethanol), sample buffer salts, or DTT in your samples. If the proteins are over-reduced, they can be negatively charged and actually repel each other across the lanes. The bands get narrower as they progress down the gel as the proteins repel each other.

Answer Id: E3834

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Can you summarize the sulfhydryl quantitation protocol for Thermo Scientific™ Ellman's Reagent?

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Sulfhydryl groups may be estimated in a sample by comparison to a standard curve composed of known concentrations of a sulfhydryl-containing compound such as cysteine, preferrably at a working range of 0.1 - 1.0 mM. A volume of 250 μl is required for the sample and standard, mixed with Ellman's Reagent Solution and Reaction Buffer, then incubated for 15 minutes at room temperature. Absorbance is read at 412 nm.

Answer Id: E13372

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Does the iBlot™ Dry Blotting System work with native or native-blue gels?

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Yes, please take a look at the Western Blotting NativePAGE™ Invitrogen™ Bis-Tris Gels Using the iBlot™ 7-Minute Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/pdf.par.18870.file.dat/native-with-iblot-app-note-v3.pdf).

Answer Id: E10755

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What species do the TaqMan™ MicroRNA Assays detect?

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Answer

Our Content is now aligned with release 20 of the miRBase database and provides comprehensive coverage for the 206 species listed. For the most up to date content, use our MicroRNA Assay Search Tool at www.thermofisher.com/taqmanmirna

Answer Id: E2764

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Why is Coomassie G-250 used as the tracking dye in NuPAGE™ LDS Sample Buffer instead of bromophenol blue?

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Answer

Coomassie G-250 will give a sharp dye front with both NuPAGE™ MES and MOPS Running Buffers and is therefore used as the tracking dye in the NuPAGE™ LDS Sample Buffer.

Bromophenol blue runs more slowly than some peptides with the NuPAGE™ MES Running Buffer system.

Coomassie G-250 migrates much closer to the moving ion front than bromophenol blue, ensuring that small peptides will not be run too far (e.g., off the gel).

Answer Id: E3835

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What other materials are required for the sulfhydryl group estimation assay that uses Ellman's Reagent?

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Answer

Here are the required materials:

-Reaction Buffer: 0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA
-Cysteine Hydrochloride Monohydrate, M.W. = 175.6: Thermo Scientific™ Cat. No. 44889
-Ellman's Reagent Solution: Dissolve 4 mg Ellman's Reagent in 1 mL of Reaction Buffer
-Spectrophotometer to read at 412 nm
-Cuvettes and test tubes for 3 mL volume

Answer Id: E13373

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Why is using the BLOCK-iT™ Fluorescent Oligo a better control than simply labeling a custom siRNA?

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The Fluorescent Oligo is labeled and modified with proprietary chemistry so that it gives a stronger and more persistent signal than other labeled siRNA might have. And you can easily assess the delivery efficiency since the fluorescence will be primarily concentrated in the cell nuclei.

Answer Id: E4404

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Should I use labeled or unlabeled siRNA for my in vivo siRNA experiments?

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We have demonstrated that labeling Stealth and Silencer™ siRNA does not hamper their knockdown potency. An alternative approach is to mix unlabeled duplexes with labeled control duplexes; this method is more commonly used with in vivo siRNA, and allows progression to clinical research unhindered by questions about the possible effects of a label.

Answer Id: E9929

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Why is more input amount required for the 100 format arrays (miRNA 3.0 Arrays)?

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Answer

To maintain comparability to previous generation arrays, a minimum of 130 ng input is recommended for 100 format arrays.

Answer Id: E14050

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I am having problems with my standard curve in the Easy-Titer Antibody Assay. What do I need to do?

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Answer

Make sure that you are mixing the samples vigorously. We set our shaker near the highest setting for this step. For best results, the beads must be fully dispersed.

Answer Id: E8630

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What is the first step in an experiment with the Gateway™ system?

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Answer

The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway™ Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway™ compatible TOPO™ Cloning vectors such as pCR™8/GW/TOPO™ and pENTR™/D-TOPO™. The final option is to use restriction enzymes to clone into a pENTR™ Dual Selection vector.

Answer Id: E3208

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What is the difference between your NativePAGE™ Bis-Tris gels and NuPAGE™ Bis-Tris gels? Can I use the NativePAGE™ buffers to run native applications with the NuPAGE™ Bis-Tris gels?

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Answer

Although the gels share some major chemical components, they are not interchangeable. NuPAGE™ Bis-Tris gels are optimized for denaturing and reducing conditions, and their neutral pH makes it difficult to use them for native applications as most proteins will have no charge or positive charge. Therefore, native applications with these gels are not recommended. NativePAGE™ Bis-Tris gels on the other hand have a different formulation that has been optimized for native electrophoresis with highest resolution. The buffers for the two types of gels should not be interchanged.

Answer Id: E10756

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