Documents & Support

1 - 15 of 96 results

What downstream applications is the aqueous buffer compatible with when using the P-PER Plant Protein Extraction reagent? Product FAQ

Answer

BCA Reducing Agent Compatible Protein Assay (Cat. No.s 23250, 23252), 1-D gel electrophoresis, 2-D gel electrophoresis (dilute sample 1:10 in sample buffer), Western blot, immunoprecipitation, ion exchange chromatography, polyhistidine-tag protein purification, and activity assays.

Answer Id:: E8185

Was this answer helpful?

Yes No

Thank you for your response

A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Citations & References

  • Authors: Gong J, Traganos F, Darzynkiewicz Z
  • Journal: Anal Biochem
  • PubMed ID: 8074286
Catalog #

Capillary gel affinity electrophoresis of DNA fragments. Citations & References

  • Authors: Guttman A, Cooke N
  • Journal: Anal Chem
  • PubMed ID: 1750704
Catalog #
  • E3565(Discontinued)
  • E1305(Discontinued)

Do any gel types require fixing before staining with the GelCode Blue Stain Reagent? Product FAQ

Answer

Yes. For gels electrophoresed with MOPS or MES running buffers, fix gel with 50% methanol and 7% acetic acid for 15 minutes and extensively wash with water.

Answer Id:: E8366

Was this answer helpful?

Yes No

Thank you for your response

How can I perform RNA electrophoresis? Product FAQ

Answer

For nondenaturing RNA electrophoresis, we recommend using our E-Gel Precast Agarose Gels. Please note that E-Gel Agarose Gels are not validated to be RNAse-free. However, many of our customers routinely use E-Gel Agarose Gels for RNA analysis with success. If RNA is run on an E-Gel Agarose Gel, any loading buffer that would be used for nondenaturing RNA electrophoresis should be fine.

For denaturing RNA electrophoresis, there are several denaturing agents to choose from, including formaldehyde, glyoxal, formamide, and methyl mercury. Denaturing conditions disrupt hydrogen bonding so that RNA runs without secondary structure, as single-stranded molecules.

For denaturing RNA electrophoresis, our E-Gel EX Agarose Gels can be used. The only denaturing agent that is compatible with the E-Gel EX system is formamide, 50-90%. Using other denaturing agents will result in poor band separation and morphology. Please note that we do not recommend running samples prepared in RNA loading buffer on the same gel with samples prepared in water. Please see below for the RNA loading buffer recipe and denaturing electrophoresis conditions:

RNA Loading Buffer:
Deionized formamide: 200 µL
10X MOPS-EDTA-Sodium Acetate Buffer (0.4 M MOPS, pH 7.0, 0.1 M sodium acetate, 10 mM EDTA): 40 µL
Deionized formaldehyde: 76 µL
Water: 14 µL

Denaturing Electrophoresis Conditions:
1. Mix 15 µL of RNA loading buffer with 1-5 µL of RNA (1-5 µg).
2. Heat samples at 65 degrees C for 10 min to denature RNA.
3. Place samples on ice immediately after heating.
4. Load entire sample onto an E-Gel EX agarose gel.
5. Electrophorese for 30 minutes.

For denaturing RNA electrophoresis under formaldehyde-free conditions, we recommend using our NorthernMax-Gly Kit (Cat. No. AM1946). With this kit, RNA samples are denatured in glyoxal/DMSO loading buffer and run on a glyoxal-containing agarose gel.

Answer Id:: E7957

Was this answer helpful?

Yes No

Thank you for your response

What is the composition of the NuPAGE LDS Sample Buffer and what is the recipe for the 4X formulation? Product FAQ

Answer

The composition of the 1X NuPAGE LDS Sample Buffer is as follows:

141 mM Tris base
106 mM Tris HCl
2% LDS
10% Glycerol
0.51 mM EDTA
0.22 mM SERVA Blue G
0.175 mM Phenol Red
pH 8.5


To prepare 10 mL of 4 X NuPAGE LDS Sample Buffer, dissolve the following reagents to 8 mL ultrapure water:

Tris HCl 0.666 g
Tris Base 0.682 g
LDS 0.800 g
EDTA 0.006 g
Glycerol 4 g
SERVA Blue G (1 solution) 0.75 mL
Phenol Red (1 solution) 0.25 mL

Mix well and adjust the volume to 10 mL with ultrapure water. Store at +4. The buffer is stable for 6 months when stored at +4°C.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

Answer Id:: E4272

Was this answer helpful?

Yes No

Thank you for your response

Epidemiological study of a food-borne outbreak of enterotoxigenic Escherichia coli O25:NM by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis. Citations & References

  • Authors: Mitsuda T; Muto T; Yamada M; Kobayashi N; Toba M; Aihara Y; Ito A; Yokota S
  • Journal: Journal of Clinical Microbiology
Catalog #

What are the fluorescent protein assays you offer and how do they differ? Which one should I choose for my samples? Product FAQ

Answer
  • The Quant-iT and Qubit protein assays are easy and accurate assays for the quantitation of protein samples ranging from 12.5 µg⁄mL to 5 mg⁄mL. These assays are highly selective for protein and exhibit very little protein-to-protein variation. Common contaminants, such as salts, solvents, or DNA (but not detergents) are well tolerated in these assays. The Qubit Protein Assay Kit is designed specifically for use with the Qubit Fluorometers, while the Quant-iT Protein Assay is designed to be used both on the Qubit fluorometer or other fluorometers and fluorescence plate readers. 
  • The NanoOrange Protein Quantitation Kit is a very sensitive and easy assay for protein quantitation, with detection as low as 10 ng/mL of protein in solution with a useful assay range of 10 ng/mL to 10 µg/mL. Common contaminants, such as salts, solvents, or DNA (but not detergents) are well tolerated in these assays. This fluorescent dye is suitable for use with spectrofluorometers and microplate readers. 
  • The CBQCA Protein Quantitation Kit is a very sensitive assay for protein quantitation, with detection as low as 10 ng/mL of protein in solution with a useful assay range of 10 ng/mL to 150 µg/mL. CBQCA covalently modifies glutamine, asparagine and primary amines and requires cyanide for the reaction. This assay is tolerant of detergents, but amines, ammonium ions and reducing agents should be avoided. CBQCA is better suited for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides.
  • The EZQ Protein Quantitation Kit provides accurate quantitation of protein samples in the range of 20 µg/mL to 5 mg/mL. The assay can be performed in the presence of detergents, urea, reducing agents, salts, solvents, dyes, and most other contaminants and is generally intended for samples prepared for 1D and 2D gel electrophoresis. 1 µL of each sample is spotted onto filter paper placed inside a specially-designed 96-well microplate, contaminants are washed away, and then the remaining bound proteins are stained with our proprietary fluorescent dye. The paper is then analyzed on a microplate reader or a laser scanner.
  • All the above assay kits come with either concentrated assay reagent and dilution buffer or a pre-diluted quantitation reagent and protein standards. The EZQ Protein Quantitation Kit also comes with a specially-designed 96-well microplate and filter paper that fits inside this microplate.

    Answer Id:: E15566

    Was this answer helpful?

    Yes No

    Thank you for your response

    Where do I find buffer recipes for Invitrogen Invitrogen gels? Product FAQ

    Answer

    The formulations of buffers for Invitrogen gels can be found in the Invitrogen Pre-Case Gel Electrophoresis Guide at https://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf online.

    Answer Id:: E4465

    Was this answer helpful?

    Yes No

    Thank you for your response

    Do you have a protocol for RNA electrophoresis on your E-Gel agarose gels? Product FAQ

    Answer

    Our E-Gel agarose gels can be used for either DNA or RNA separation. RNA separation occurs under non-denaturing or denaturing conditions. Please note, our gels are not QC tested for the presence of RNases. See our suggestions below for running your non-denaturing or denaturing samples:

    Non-denaturing conditions
    1. Mix RNA sample with 15 µL of RNase-free water.
    2. Do not heat. Load the entire sample onto the E-Gel agarose gel.
    3. Electrophorese for 30 min.

    Denaturing conditions
    1. Mix 15 µL of RNA loading buffer with 1-5 µL of RNA (1-5 µg).
    2. Heat samples at 65 degrees C for 10 min to denature RNA.
    3. Place samples on ice immediately after heating.
    4. Load entire sample onto E-Gel agarose gel.
    5. Electrophorese for 30 min.

    Note:
    - The only denaturing agent that is compatible with the E-Gel system is formamide, 50-95%. Heating the sample for 5 min at 65 degrees C should be sufficient for denaturing. Using other denaturing agents like glyoxal, formaldehyde, or urea will result in very poor separation and band morphology on E-Gel agarose gels.
    - Additionally, we do not recommend running samples with RNA loading buffer on the same gel as samples loaded with water.
    - E-Gel EX Double Comb agarose gels and E-Gel Double Comb agarose gels with SYBR Safe stain have not been tested for use in RNA applications.

    Answer Id:: E7965

    Was this answer helpful?

    Yes No

    Thank you for your response

    What does the NuPAGE Antioxidant do? How much do I add to the NuPAGE running buffer? Product FAQ

    Answer

    The reducing agents DTT and beta-mercaptoethanol will not migrate through the gel with the sample in the neutral pH environment of the NuPAGE Gels. Instead, the reducing agent tends to remain at the top of the gel. Disulfide bonds are less reactive at neutral pH and are less likely to reoxidize than in a higher-pH system. However, in the absence of an antioxidant some reoxidization may occur during the electrophoresis, resulting in slightly diffuse bands.

    The NuPAGE Antioxidant (a proprietary reagent) is added to the running buffer in the upper (cathode) buffer chamber only when performing electrophoresis under reducing conditions. The NuPAGE Antioxidant migrates with the proteins during electrophoresis preventing the proteins from reoxidizing and maintaining the proteins in a reduced state. The NuPAGE Antioxidant also protects sensitive amino acids such as methionine and tryptophan from oxidizing.

    We also recommend using the NuPAGE Antioxidant with reduced samples that have been alkylated, for optimal results.

    The NuPAGE Antioxidant is NOT compatible with gel systems other than the NuPAGE system as the antioxidant is not efficient at higher pHs of other gel systems.

    0.5 mL of antioxidant is added to 200 mL (400x dilution) of running buffer and placed in the upper buffer chamber.

    Answer Id:: E3837

    Was this answer helpful?

    Yes No

    Thank you for your response

    Can I prepare my protein sample with the reducing agent and store it for future use? Product FAQ

    Answer

    DTT is not stable, so it must be added and the reduction performed just prior to loading your samples.

    Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

    Answer Id:: E6382

    Was this answer helpful?

    Yes No

    Thank you for your response

    Is there a fixation step in the Thermo Scientific 6xHis Protein Tag Stain Reagent Set staining procedure? Product FAQ

    Answer

    There is no fixation step in the Thermo Scientific 6xHis Protein Tag Stain Reagent Set staining procedure. Hence, staining with this kit does not inhibit subsequent total protein staining with general protein stains, or electrophoretic transfer to membrane. Note: Bis-Tris gels run in MOPS or MES buffer may require fixing in 50% methanol: 7% acetic acid for 15 minutes before performing the stain procedure. After electrophoresis, fix the gel and then proceed with Step 1 of the procedure.

    Answer Id:: E11194

    Was this answer helpful?

    Yes No

    Thank you for your response

    With the NorthernMax Kit, I am seeing high background on the blot, but not in the lanes? Why is this? Product FAQ

    Answer

    Here are possible causes and solutions:
    - An incompatible or low-quality membrane may have been used. Positively charged nylon membrane such as Ambion BrightStar-Plus Membrane is strongly recommended, particularly for non-isotopic Northerns. Nitrocellulose membranes are not compatible with the NorthernMax Transfer Buffer and should not be used with this kit.
    - The membrane could have dried out during the procedure. Do not allow the membrane to dry out at any time between pre-hybridization and exposure. If the membrane becomes dry in the hybridization or washing steps, or during the non-isotopic detection procedure, severe background will often result.
    - The reagents were not evenly distributed. Do not add probe directly to the membrane and pre-hybridization solution; dilute it in ~1 mL of ULTRAhyb Buffer immediately before adding it to the pre-hybridization buffer. Be sure that all solutions are free to move over the entire surface of the membrane during each step in the procedure and use gentle agitation of the membranes for each incubation. If necessary, increase volumes or switch to another container. If treating more than one membrane at a time, be sure they do not stick together. Make sure there are no folds, creases, or bubbles present if hybridizing in plastic bags.
    - ULTRAhyb Ultrasensitive Hybridization Buffer may not have been completely solubilized. ULTRAhyb Buffer should be heated to 68 degrees C for 15-30 min before it is added to the blot for pre-hybridization to completely solubilize all components.
    - The reagents may have been contaminated by microbes. This is especially problematic if blocking buffers used in non-isotopic detection systems become contaminated with fungi or bacteria. Follow the manufacturer's recommendations for use and storage of reagents. Replace any reagents that appear contaminated (cloudy, filmy, overly viscous, etc.: not due to precipitation).
    - Particulate matter may have been deposited on the membrane. Be sure to handle membranes only by the edges using powder-free or rinsed gloves and forceps. Protect wet membranes from coming in contact with dust (i.e., the floor). Store membranes in a clean environment at all times.
    - Precipitates may have been present in the non-isotopic detection reagents. Follow manufacturer's recommendations for filtration or centrifugation of reagents, particularly blocking buffer and secondary detection reagents.
    - Agarose or Transfer Buffer may have dried on the membrane. Rinse membrane briefly in 1X Gel Running buffer (it is okay to use buffer from the electrophoresis chamber) after transfer and before crosslinking. - The film may have been exposed to static charges during development. Wipe the plastic film covering the membrane with a damp tissue and allow to air dry before applying film.
    - The blot may have been too wet when exposed to the film. Blot membrane briefly on filter paper until it is damp but not dripping. Wrap immediately in plastic and expose to film. There should not be any moisture on the outside of the plastic covering the membrane when the film is applied. Carefully blot dry any liquid that seeps out of the edges of the plastic wrap.

    Answer Id:: E19242

    Was this answer helpful?

    Yes No

    Thank you for your response

    With the NorthernMax-Gly Kit, I am seeing high background on the blot, but not in the lanes? Why is this? Product FAQ

    Answer

    Here are possible causes and solutions:
    - An incompatible or low-quality membrane may have been used. Positively charged nylon membrane such as Ambion BrightStar-Plus Membrane is strongly recommended, particularly for non-isotopic Northerns. Nitrocellulose membranes are not compatible with the NorthernMax Transfer Buffer and should not be used with this kit.
    - The membrane could have dried out during the procedure. Do not allow the membrane to dry out at any time between pre-hybridization and exposure. If the membrane becomes dry in the hybridization or washing steps, or during the non-isotopic detection procedure, severe background will often result.
    - The reagents were not evenly distributed. Do not add probe directly to the membrane and pre-hybridization solution; dilute it in ~1 mL of ULTRAhyb Buffer immediately before adding it to the pre-hybridization buffer. Be sure that all solutions are free to move over the entire surface of the membrane during each step in the procedure and use gentle agitation of the membranes for each incubation. If necessary, increase volumes or switch to another container. If treating more than one membrane at a time, be sure they do not stick together. Make sure there are no folds, creases, or bubbles present if hybridizing in plastic bags.
    - ULTRAhyb Ultrasensitive Hybridization Buffer may not have been completely solubilized. ULTRAhyb Buffer should be heated to 68 degrees C for 15-30 min before it is added to the blot for pre-hybridization to completely solubilize all components.
    - The reagents may have been contaminated by microbes. This is especially problematic if blocking buffers used in non-isotopic detection systems become contaminated with fungi or bacteria. Follow the manufacturer's recommendations for use and storage of reagents. Replace any reagents that appear contaminated (cloudy, filmy, overly viscous, etc.: not due to precipitation).
    - Particulate matter may have been deposited on the membrane. Be sure to handle membranes only by the edges using powder-free or rinsed gloves and forceps. Protect wet membranes from coming in contact with dust (i.e., the floor). Store membranes in a clean environment at all times.
    - Precipitates may have been present in the non-isotopic detection reagents. Follow manufacturer's recommendations for filtration or centrifugation of reagents, particularly blocking buffer and secondary detection reagents.
    - Agarose or Transfer Buffer may have dried on the membrane. Rinse membrane briefly in 1X Gel Running buffer (it is okay to use buffer from the electrophoresis chamber) after transfer and before crosslinking.
    - The film may have been exposed to static charges during development. Wipe the plastic film covering the membrane with a damp tissue and allow to air dry before applying film.
    - The blot may have been too wet when exposed to the film. Blot membrane briefly on filter paper until it is damp but not dripping. Wrap immediately in plastic and expose to film. There should not be any moisture on the outside of the plastic covering the membrane when the film is applied. Carefully blot dry any liquid that seeps out of the edges of the plastic wrap.

    Answer Id:: E19254

    Was this answer helpful?

    Yes No

    Thank you for your response

    Results per page
    spinner