How should I prepare my membranes for WesternBreeze™ Chemiluminescent detection?

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For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternBreeze™ Chemiluminescent Immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternBreeze™ Chemiluminescent Immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 mins before proceeding to the WesternBreeze ™ Chemiluminescent Immunodetection protocol.

For Native-PAGE western blot, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 mins before proceeding to the WesternBreeze™ Chemiluminescent Immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternBreeze™ Chemiluminescent Immunodetection protocol.

Answer Id: E10837

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Can I purchase the Chemiluminescent Substrate and Chemiluminescent Substrate Enhancer from the WesternBreeze™ Chemiluminescent Detection Kits as standalone products?

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Answer

Yes, you can purchase them as standalone products and below are the Cat. Nos.:

- Chemiluminescent Substrate, Cat. No. WP20002
- Chemiluminescent Substrate Enhancer, Cat. No. WP20003

Answer Id: E10838

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What reagents inhibit SuperScript™ II RT activity?

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Answer

The following reagents will inhibit SuperScript™ II RT activity by at least 50%.
34% glycerol
4 μg/mL heparin
0.0025% (w/v) SDS
5% (v/v) formamide
17.0% (v/v) DMSO
4 mg/mL glycogen
30 mM guanidine-HCl
15 mM guanidine isothiocyanate
1 mM EDTA
2.5 mM NaPPi
0.4 mM Spermidine

Answer Id: E2959

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If there is a precipitate in my culture and the pH of the media has changed dramatically (+/-1 pH unit), is there a problem?

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You most likely have bacterial or fungal contamination. You should discard the culture.

Answer Id: E3964

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Can I use the SuperScript™ IV RT Reaction Buffer with SuperScript™ II RT or SuperScript™ III RT enzymes?

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No, we do not recommend using SuperScript™ IV RT Reaction Buffer with the SuperScript™ II RT or SuperScript™ III RT enzymes. For optimal performance, we recommended using the matching reaction buffer for each enzyme.

Answer Id: E13454

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What are the Invitrogen™ AP Chemiluminescent Substrate (CDP-Star™ substrate), Invitrogen™ AP Chemiluminescent Substrate Enhancer (Nitro Block II™ enhancer), and Invitrogen™ ECL Chemiluminescent Substrate?

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Invitrogen™ AP Chemiluminescent Substrate (CDP-Star™ substrate) and Invitrogen™ ECL Chemiluminescent Substrate are non-radioactive substrates for chemiluminescence-based immunodetection of alkaline phosphatase (AP) or horse radish peroxidase (HRP), respectively, on western blots or dot blots. Invitrogen™ Chemiluminescent Substrates provide detection sensitivities superior to that of precipitating chromogenic substrates. Low picogram levels of detection can be achieved using either X-ray film or imaging equipment.

Invitrogen™ AP Chemiluminescent Substrate consists of a ready-to-use solution of CDP-Star™ substrate, a dioxetane-based substrate for detection of alkaline phosphatase. The Invitrogen™ AP Chemiluminescent Substrate Enhancer consists of a 20X solution of Nitro-Block-II™ enhancer that is mixed with the AP Chemiluminescent Substrate when probing blots on nitrocellulose membranes. Do not use the enhancer with PVDF membranes, as it can produce high background. Light emission for this substrate builds rapidly in the first 20 mins after membrane incubation and reaches peak intensity after 45-60 mins. Light emission has a maximal wavelength at 461-466 nm, and continues for several hours and in some cases, for days. Invitrogen™ AP Chemiluminescent Substrate and Invitrogen™ AP Chemiluminescent Substrate Enhancer are available as standalone products (Cat. Nos. WP20002 and WP20003) and are also components of the WesternBreeze™ Chemiluminescent Detection kits.

Invitrogen™ ECL Chemiluminescent Substrate Reagent Kit is a two-part reagent consisting of a luminol and an enhancer for the detection of horse radish peroxidase. Reagent A and Reagent B are mixed in equal volumes before application to the blot. Light emission for Invitrogen™ ECL Chemiluminescent Substrate is most intense from 5-30 minutes after membrane incubation and decreases slowly with time over the course of several hours.

Answer Id: E10839

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What are the recommended conditions for proteinase K treatment when isolating RNA or DNA samples?

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Answer

Concentration: Generally proteinase K is used in the concentration range of 50 to 500 μg/mL at 65 degrees C in the presence of SDS (0.5-1%).

Temperature optimum: 65 degrees C; 12X more active at 65 degrees C than at 25 degrees C.

pH: Proteinase K is stable over a wide pH range (4.0 to 12.5), with optimal activity at pH 6.5 to 9.5. It is most stable at pH 8.
Inactivation: Heat inactivate Proteinase K at 80 degrees C for 15 min. Phenol extraction is the recommended method to ensure complete inactivation.

Inhibited by: PMSF (0.1 to 1.0 mM of PMSF is usually sufficient for inhibition of Proteinase K)

Not inhibited by: Proteinase K is not inactivated by metal ions, chelating agents (e.g., EDTA), sulfhydryl reagents or by trypsin or chymotrypsin inhibitors. Activity can be stimulated by addition of denaturing agents (SDS and urea).

Answer Id: E2960

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How do I increase the signal in immunodetection methods (such as ELISA, electrophoretic transfer, membrane-bound immunodetection, immunocytochemistry, immunofluorescence microscopy, and flow cytometry)?

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Answer

In general, no signal or weak signal can be a result of one or more of the following:

(1) Reagents were omitted or added in an incorrect order. Use all reagents in the proper sequence.

(2) Incorrect reagents were used. Use matched reagents (for example, a mouse primary antibody with an anti-mouse secondary antibody).

(3) Insufficient amounts of antigen were present, which prevented detection with the immunodetection procedure employed. You could:
-Use a more sensitive detection system.
-Use a secondary antibody instead of a directly labeled primary antibody. This may increase the signal 10-fold or more.
-Increase the primary antibody concentration.
-Increase the incubation time of the primary antibody with the antigen.
-Use more antigen.

(4) Improper storage of reagents resulted in degradation. Store reagents at recommended conditions.

(5) Low-affinity primary antibody was lost during immunodetection procedure. Try higher affinity antibody if available. Increase the incubation time or concentration of primary antibody with the antigen to maximize the amount of primary antibody bound. Decrease wash volumes and/or times to decrease dissociation of the primary antibody.

(6) Primary antibody reacted poorly with denatured antigen. Use procedure and buffers that allow retention of the native form of the antigen.

(7) Incubation times with secondary antibody or the streptavidin or avidin conjugate were insufficient. Increase these incubation times.

(8) Incorrect incubation temperatures were used in one or more of the steps. Use recommended incubation temperatures.

(9) Horseradish peroxidase conjugate activity was inhibited. Do not use sodium azide in the conjugate dilution buffer and the substrate buffer. Use the recommended concentration of hydrogen peroxide. The resulting signal will be reduced if either insufficient or excessive amounts are employed.

Answer Id: E3965

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What is the NetAffx™ Analysis Center?

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Answer

The NetAffx™ Analysis Center is the most comprehensive resource of integrated array contents and functional annotations available. The flexible query capabilities provided help you retrieve biological information for specific probe sets. Please read our Analysis Center tutorial for more information (http://www.affymetrix.com/analysis/index.affx#1_1).

Answer Id: E13455

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What types of inhibitors are present to help preserve any post-translational modifications of the expressed proteins on the Human ProtoArrays™ microarray? What tests have been done to confirm that post-translational modifications are still present on the spotted proteins?

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Answer

Protease inhibitors are used during the protein purification steps. All the proteins are expressed and purified at 4 degrees C. The protein arrays are manufactured at 4 degrees C and stored immediately at -20 degrees C. Phosphospecific antibody profiling experiments have shown that the proteins retain their posttranslational modifications.

Answer Id: E4552

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What is the recommended temperature for hybridization for Affymetrix™ miRNA Arrays?

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Answer

The following hybridization temperatures should be used with each version of Affymetrix™ miRNA Array:

- GeneChip™ miRNA Arrays 48 degrees C

- Affymetrix™ miRNA Array Plates 48 degrees C

- Affymetrix™ miRNA Array Strips 48 degrees C

Answer Id: E14132

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Can poly(A)+ mRNA be isolated directly from cell lysates using the MagJET mRNA Enrichment Kit?

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Answer

No, the kit is suitable for mRNA isolation from previously purified total RNA samples.

Answer Id: E8717

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Are the TA or TOPO™ TA Cloning™ vectors available to purchase without competent cells? What about your Zero Background™, Zero Blunt™, or Zero Blunt™ TOPO™ Cloning Kits

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Answer

Most of our cloning vectors are offered in a complete format, which includes competent cells. While in most cases other cells can be used with our vectors, we cannot guarantee the results you will get with our cloning vectors if you use your own competent cells. For this reason, most TOPO™ TA Cloning™ vectors can only purchased in a complete kit with cells, although there are a few exceptions. Non-TOPO™ vectors are generally available in multiple formats, with and without cells. To get the most current information on available products, visit the Cloning section of our website under Products & Service.

Zero Background™ and Zero Blunt™ vectors are available without competent cells provided, but you should especially careful in choosing competent cells to use with them. These vectors contain the ccdB gene for efficient negative selection of clones without insert, and some E. coli strains are not compatible with the mechanism of negative selection by the lethal activity of the ccdB gene product. In particular, cells with the F' episome have a ccd locus containing the ccdA gene, which prevents ccdB protein cell-killing. Therefore, cells without the F' episome are recommended so that only the CcdB protein will be expressed, and its cell-killing ability will not be inhibited or reduced by CcdA.

Answer Id: E3331

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What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?

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Answer

Successful amplification of long PCR targets is dependent on variables such as sufficient extension time during the PCR amplification, cosolvent addition, pH of the reaction buffer, salt concentration, primer design, use of a hot start, DNA sample integrity, and the enzyme's proofreading and polymerase activities. A few examples of our long PCR enzymes include our Elonagase enzyme mix that can be used for amplicons up to 30kb (blend of Taq and proofreading enzyme) or our Phire Hot Start II enzyme mix that can be used for amplicons up to 20 kb (Taq polymerase). Read more here: https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/long-fragment-pcr.html

Answer Id: E1083

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What are the applications and recommended protocols for Topoisomerase I?

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Answer

Topoisomerase I has two general applications: to relax supercoiled DNA, and to generate samples with defined superhelical density. Generating samples with defined superhelical density is described in Analytical Biochemistry, v.122, p. 253, by Singleton and Wells (1982).

For relaxing supercoiled DNA, use the following protocol:

1. Add 0.5 ?g supercoiled PhiX 174 DNA, 50 mM TrisHCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA, 30 ?g/ml BSA, and 1 unit Topoisomerase I to a final volume of 50 ?l.
2. Incubate 30 min at 37°C.
3. Analyze the treated samples on a 1% gel that does not contain ethidium bromide. Stain with ethidium bromide after electrophoresis is complete.

Relaxed (form II) DNA will migrate slower than supercoiled (form I) DNA. If supercoiled DNA other than PhiX 174 is used, the amount of enzyme must be adjusted. As a general guideline, use up to 4 units of enzyme to relax 0.25 ?g of pUC DNA.

Answer Id: E2962

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