Regulatory requirements vary by product. Please see product-specific literature to determine product use.

Product FAQ

Can I use ethanol instead of methanol in the NuPAGE transfer buffer (and in transfer buffers in general)?

Answer

We find that including ethanol in the transfer buffer is just as effective as including methanol. You may use either of these alcohols at an equivalent concentration.

Answer Id: E3278

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Product FAQ

How much can the pH of the Western transfer buffer vary and still be effective?

Answer

If the buffer deviates from its expected pH by 0.2 units or higher, discard and remake the buffer after rechecking the reagents and the water purity. Do not adjust the pH with acid or base, as this will increase the conductivity of the buffer and result in higher current during the transfer.

Answer Id: E3279

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Product FAQ

What is the biotin-binding capacity for UltraLink Biosupport Medium?

Answer

The biotin-binding capacity is >3 mg biotinylated BSA/ml of resin, or >120 nMoles biotin/mL of resin which is equilvalent to approximately 30 µg of biotin or 30 units of biotin/ml.

Answer Id: E8256

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Product FAQ

I am looking for hepatocytes with particular characteristics. Who should I contact?

Answer

Our hepatocytes are prequalified and sold according to application, such as induction, short-term metabolism, and transporter uptake. If you require cells with particular P450 values or donor specifications, please contact our Hepatic Biology Tech Support Team at hepaticproducts@invitrogen.com to help with specific lot recommendations.

Answer Id: E16556

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Product FAQ

When blotting with the XCell II Blot Module, what would happen if I filled the outer buffer chamber with transfer buffer instead of water?

Answer

This is perfectly acceptable with the XCell II Blot Module. The liquid in the outer buffer chamber only serves as a coolant or heat sink. The reason why water is recommended is because it is a less expensive alternative.

Answer Id: E3280

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Product FAQ

I’m seeing PCR products in the minus-RT control after performing my Cells-to-CT experiment. What does this mean?

Answer

If PCR products are seen in the minus-RT control reaction, but not in the no-template control, it indicates that genomic DNA remains in the sample and that genomic DNA was amplified in real-time PCR. Please follow the suggestions below:

- Ensure the DNase I is mixed thoroughly into the Lysis Solution.
- Use fewer cells per lysis reaction.
- Lyse cells using Lysis Solution that is at room temperature, and make sure that the lysis reaction occurs at room temperature.
You can also try increasing the incubation time of the lysis reaction to 8 minutes and/or using Lysis Solution that has been warmed up to 25 degrees C for cell lysis.

Answer Id: E7882

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Product FAQ

When should the UltraLink Immobilized Streptavidin Plus Support be used in place of streptavidin immobilized on agarose?

Answer

UltraLink Immobilized Streptavidin Plus Support should be used when you are planning to perform anything other than gravity filtration with a column. Streptavidin Plus UltraLink Resin is often preferred for immunoprecipitation because the white appearance of the support allows the pellet to be visualized more easily than the translucent pellet of agarose supports.

Answer Id: E8257

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Product FAQ

What should I plate hepatocyte cells on?

Answer

Hepatocytes need to be plated on collagen I-coated culture vessels.

Answer Id: E16557

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Product FAQ

Are IPTG and X-gal necessary for blue/white screening of colonies?

Answer

With a vector containing the lac promoter and the LacZ alpha fragment (for alpha-complementation), blue/white screening can be used as a tool to select for presence of an insert. X-gal is added to the plate as a substrate for the LacZ-encoded beta-galactosidase enzyme and must always be present to see the blue color in the blue/white screening.

In cases where the lacIq repressor is present (either provided by the host cells on an F' episome, i.e. TOP10F', or expressed from the plasmid), it will repress expression of LacZ from the lac promoter and prevent the formation of the blue color with X-gal. This repression can be reversed by adding IPTG to the media in addition to X-gal, which will inhibit the action of lacIq and re-activate expression from the lac promoter.

Answer Id: E3322

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Product FAQ

Do you have any tips to improve results when Western transferring two gels at the same time?

Answer

Less-than-optimal transfer in the second gel is not uncommon and frequently requires adjustments in protocol. To achieve similar transfer efficiencies in the two gels you can transfer them one at a time or consider the following suggestions:

Make sure that the blot module is not overfilled with buffer, i.e., fill to point where pads are just covered and no higher. If the buffer level is too high, some current will bypass the gels. If it is overfilled, shorting, arcing, and other problems can occur.

Use antioxidant when transferring reduced proteins.

Increase transfer time by 30 to 60 minutes. The longer run time allows the slower moving proteins time to move out of the gel.

As proteins are being driven out of the gel by the SDS on their surface, it is important that enough is retained to keep them mobile. However, the methanol that is included in the transfer buffer will remove some of the SDS, and the higher the methanol concentration, the more SDS is washed off of the protein. Methanol is included in order to increase binding to nitrocellulose membranes via hydrophobic interactions. However, if using PVDF or nylon membranes, less or even no methanol may be required. 20% methanol is a good starting point for most situations In the event that the transfer is less than ideal, methanol levels should be reduced by at least 50%. The optimal percentage should be determined empirically. Something to keep in mind is that with increased mobility, the proteins may just move straight though the first membrane. In this case, a second membrane may trap these samples.

It is possible to BRIEFLY (1 min) soak the second gel in transfer buffer with 0.01 to 0.02% SDS before assembling the sandwich. This must be a short soak or the proteins will tend to diffuse out of the gel. Adding 0.01-0.02% SDS to the transfer buffer and using it for the transfer will also aid in protein mobility.

Halfway through the transfer process (30 min), swap the front gel and back gel in the blotter. This is fairly easy to do if you just swap pad-paper-gel-membrane-pad sections.

Another recommendation: if you must transfer simultaneously, and want to be able to compare results of both blots, use an internal control in both gels to get an idea of the relative transfer efficiency and to afford some normalization of the results.

Answer Id: E3281

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Product FAQ

I am interested in isolating mRNA. Can I use one of your kits to isolate mRNA directly from my starting sample or do I need to isolate total RNA and enrich for mRNA?

Answer

Please use the selection guide (http://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-types/mrna-extraction.html) to view the kits we offer for mRNA purification, as well as the differences between them.

Answer Id: E7883

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Product FAQ

How do I perform affinity purifications with conjugated SulfoLink Affinity Columns?

Answer

Equilibrate the column with 3-5 bed-volumes of an appropriate binding buffer. Add 1 mL of sample for each 2 mL column (serum should be diluted at least 1:1 with binding buffer). Add an additional 200 µl of binding buffer to ensure that the entire sample has entered the gel bed. Cap the column bottom and top. Incubate the column for 1 hour. Wash away non-bound proteins with 5-7 bed-volumes of binding buffer or 1 M NaCl. Elute the bound sample by adding small fractions (0.5-1.0 ml) of elution buffer such as Thermo Scientific IgG Elution Buffer (Cat. No. 21004).

Answer Id: E8258

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Product FAQ

I forgot to add Bluo-gal to my plates. Can I add it later?

Answer

On the day you intend to pick plaques, make a solution of Bluo-gal in DMSO at 20 mg/mL. Add 50 µL per plate and spread with a glass spreader under sterile conditions. Wait 30-60 min, and your plaques should turn blue.

Answer Id: E9471

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Product FAQ

What is HepExtend Supplement?

Answer

HepExtend Supplement (50X) is an enriched supplement designed to enhance the culture lifespan (cell viability, function, and number of days in culture) of cryopreserved primary hepatocytes. We recommend using the supplement in conjunction with William's E Medium (Cat. No. A1217601) and Hepatocyte Maintenance Supplement Pack (Cat. No. CM4000), following the recommended hepatocyte culture methods. When cultured in the presence of the HepExtend Supplement, primary human hepatocyte lots will live and be functional for 10 days or longer depending on the lot of cells and other assay conditions. HepExtend Supplement does not contain growth factors, cytokines, fetal bovine serum, or pharmaceutical small molecules that are known to interfere with primary hepatocyte function.

Answer Id: E16558

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Product FAQ

Can an insert be TA-cloned in frame with the LacZ gene in the pCRII-TOPO vector? Where is the initiation codon in the pCRII-TOPO sequence?

Answer

The first ATG is contained within the M13 Reverse Primer sequence. Note that there is an additional ATG in frame just 6 bases further down.
The reading frame is as follows: GAA, TTC, GGC, TT., ..., etc. Therefore, 1 additional base is needed 5' to the reading frame of the gene of interest in order to be in frame with the LacZ gene.

Answer Id: E3324

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