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The North2South Chemiluminescent Hybridization and Detection Kit is as sensitive as Southern and Northern blotting procedures using radioactive probes (³²P or 35S). Femtomole quantities of biotin-labeled probe can be easily detected after binding to Streptavidin-HRP with only 5 to 10 minutes of exposure to X-ray film. The sensitivity of the assay will depend on the amount of label incorporation. For instance, a probe labeled with 10 molecules of biotin or HRP per strand will produce up to 10 times more signal than a probe with only 1 molecule of biotin or HRP per strand.

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Biotin end-labeled DNA containing a putative or known binding site is incubated with a nuclear extract or purified factor. This reaction is then subjected to gel electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. Because DNA-protein complexes migrate slower than DNA alone in a native gel, a “shift” in the migration of the labeled DNA occurs. The biotin end-labeled DNA is detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate developed for the LightShift Kit. The signal is then detected with X-ray film or a high quality CCD camera.

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SuperSignal West Dura Extended Duration Substrate is a luminol-based enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate. It is oxidized to an excited state product in the presence of the stable peroxide and horseradish peroxidase. As the product decays to a lower energy state, light is released at 425 nm. The emitted light is then captured on X-ray film or by using a CCD camera.

SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled CCD camera-based detection systems. Unlike substrates with signals that decline to barely detectable levels in 30-60 min, the signal produced with SuperSignal West Dura Extended Duration Substrate is stable for 24 hr, allowing multiple film or camera exposures.

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SuperSignal West Femto Maximum Sensitivity Substrate is a luminol-based enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate. It is oxidized to an excited state product in the presence of the stable peroxide and horseradish peroxidase. As the product decays to a lower energy state, light is released at 425 nm. The emitted light is then captured on X-ray film or by using a CCD camera.
SuperSignal West Femto Maximum Sensitivity Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled CCD camera-based detection systems. Unlike substrates with signals that decline to barely detectable levels in 30-60 min, the signal produced with SuperSignal West Femto Maximum Sensitivity Substrate is stable for 24 hr, allowing multiple film or camera exposures.

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Please review the following applications for TSP (Transferable Solid Phase) :
- Reactions such as the ELISA can be performed on the TSP. The pins are coated by submerging in the analyte solution contained in a 96-well plate. Washing and reaction with succeeding antibody or streptavidin conjugates can be performed by transferring the TSP into a washing tray or second 96-well plate filled with the appropriate solution. The TSP is placed into a substrate solution until color is observed and is then removed to ensure a simultaneous start and stop to the enzymatic reaction.
- TSP is available sterile for screening hybridoma cells for the production and secretion of monoclonal antibodies.
- TSP can be used for radioimmunoassays because it can be placed directly on X-ray film and exposed for several hours. Only the tips of the pins should be incubated with the radiolabeled reagent.
- TSP can be used with the OmniTray for performing dot blots and for replicating bacterial clones from a 96-well plate.

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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.

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Here is the protocol you can follow:
(1) Run the gel. Locate the DNA of interest by autoradiography or by examination of ethidium bromide stained gels.
(2) Use a razor blade to cut out the upper 1/2 to 2/3 of the band of interest.
To recover a fragment of DNA identified by autoradiography, cut out from the X-ray film a small rectangle encompassing the image of the fragment. Align the film over the gel and cut out the segment of polyacrylamide bordered the rectangular hole in the film.
(3) Transfer the gel slice to a microcentrifuge tube. Use a sterile glass rod to crush the slice against the wall of the tube.
(4) Calculate the volume of the slice and add 1 to 2 volumes of elution buffer to the microcentrifuge tube.
Elution buffer: 0.5 M ammonium acetate, 10 mM magnesium sulfate, 1 mM EDTA (pH 8.0), 0.1% SDS
It is convenient if the volume of elution buffer is no greater than 0.5 mL since the eluted fragment of DNA can then be precipitated with ethanol in a single tube.
(5) Close the tube and incubate at 37 degrees C on a rotary platform. Small fragments of DNA (500 bases) are eluted in 3 to 4 hours.
(6) Centrifuge the sample at 12,000 g for 1 minute at 4 degrees C in a microcentrifuge. Transfer the supernatant to a fresh microcentrifuge tube, being careful to avoid transferring fragments of polyacrylamide.
(7) Add additional 0.5 mL volume of elution buffer to the pellet of polyacrylamide, vortex briefly, and re-centrifuge. Combine the two supernatants.
(8) Remove any remaining fragments of polyacrylamide by passing the supernatant through a disposable plastic column or a syringe barrel containing Whatman GF/C filter or packed siliconized glass wool.
(9) Add 2 volumes of ethanol at 4 degrees C and store the solution on the ice for 30 min. Recover the DNA by centrifugation at 12,000 g for 10 min at 4 degrees C in a microcentrifuge.
(10) Re-dissolve the DNA in 200 µL of Tris-EDTA (TE) buffer (pH 7.6), add 25 µL of 3M sodium acetate (pH 5.2) and re-precipitate the DNA with 2 volumes of ethanol as described in step 9. TE Buffer (pH 7.6): 10 mM Tris HCl (pH 7.6), 1mM EDTA (pH 8.0).
(11) Carefully rinse the pellet once with 70% ethanol and redissolve the DNA in TE buffer (pH 7.6) to a final volume of 10 µL.
(12) Check the amount and the quality of the fragment by polyacrylamide gel electrophoresis.

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Here are possible causes and solutions:
- The hybridization temperature was not optimal. Temperatures of 42 degrees C for DNA probes and 68 degrees C for RNA probes usually give excellent results in ULTRAhyb Buffer; however, hybridization conditions that are substantially above or below the optimum for a given probe can lead to reduced signals. This is most often encountered with probes having unusually high GC or AT content, probes that have a high degree of mismatch with the target, or oligonucleotide probes. Hybridization conditions for these probes may be best determined empirically. See section V.D. Determining Optimum Hybridization Temperature on page 32 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.pdf) for recommendations.
- The probe was degraded. Use radiolabeled probes quickly. The high specific activity probes used in Northerns degrade rapidly due to autoradiolysis, resulting in low signal and/or high background. Make sure non-isotopically labeled probes have not been degraded by nuclease contamination.
- The specific activity of the probe was too low. The specific activity of the probe should be at least 1x10E8 cpm/µg and preferably greater than 1x10E9 cpm/µg. If using end-labeled oligonucleotide probes, switch to a more sensitive probe type such as internally-labeled, longer probes.
- The probe concentration was too low. Calculate cpm (radiolabeled probes) or concentration by A260 (non-isotopic probes) after removal of unincorporated nucleotides. Use 1x10E6 cpm/mL for radiolabeled probes, 1 pM for non-isotopically labeled DNA probes, and 0.1 nM for non-isotopically labeled RNA probes.
- Insufficient exposure. Low copy number RNAs can take >3 days to show up with 32P-labeled probes at -80 degrees C and intensifying screens. With chemiluminescent detection systems, there is often a delay before peak light emission is reached. Low copy number RNAs often take 30 min to 1 hr to show up with the BrightStar system after an initial 2 to 4 hr delay.
- Insufficient target RNA. Load up to 30 µg total RNA. If this is not enough to give a strong signal, poly (A+) RNA should be used (mRNA represents only about 0.5-3% of total RNA). If this is still not enough, switching to a more sensitive technique such as RT-PCR should be considered.
- The message co-migrated with ribosomal RNA. Electrophoresis and/or transfer of target RNA can be hindered by the large amount of ribosomal RNA. Messages that co-migrate with ribosomal RNA may give better signals when poly (A+) selected RNA is used rather than total RNA.
- Inappropriate use of intensifying screens, and exposure temperature. Intensifying screens are only effective if they are incubated with the membrane and film at -70 degrees C. Conversely, if screens are not used, the X-ray film will be much less sensitive at -70 degrees C than at room temperature. When using two intensifying screens, the exposure setup should be as follows: blot - screen - film - screen. The radioactive energy from the blot will go through the adjacent intensifying screen, expose the film and then be reflected back and forth between the two intensifying screens.
- There could have been procedural issues such as:
-Incomplete denaturation of double-stranded probes. Double-stranded probes that are not denatured usually yield little to no hybridization signal. If the probe is only partially denatured, the effective probe concentration will be reduced. Instructions for denaturation of probes can be found in section II.F.3 on page 14 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.pdf).
-Sub-optimal transfer of RNA to membrane. See section IV.C. Problems During Transfer on page 22 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.pdf).
-Inadequate RNA crosslinking/Over-exposure to UV light. Be sure to follow recommendations for crosslinking of RNA to the membrane. If an uncalibrated hand-held UV source or transilluminator is being used for UV crosslinking, do the pilot experiment for calibrating UV sources. See Ambion Technical Bulletin #169 (https://www.thermofisher.com/us/en/home/brands/invitrogen/ambion.html) for information. Minimize exposure to UV light during UV shadowing.
-Transfer too long/too short. Transfer for 15 min per mm of gel thickness.
-Incompatible membrane. A positively charged nylon membrane, such as Ambion Bright-Star-Plus Membranes, is strongly recommended, particularly for non-isotopic Northerns. Nitrocellulose membranes are not compatible with the NorthernMax Transfer Buffer and should not be used with these kits.
-Failure to follow non-isotopic detection protocols. Follow the manufacturer's recommendations closely. Do not attempt to expose non-isotopically labeled blots to film in a freezer as with radiolabeled blots. Low temperatures will stop the enzymatic reactions and no light will be emitted.

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Here are possible causes and solutions:
- The hybridization temperature was not optimal. Temperatures of 42 degrees C for DNA probes and 68 degrees C for RNA probes usually give excellent results in ULTRAhyb Buffer; however, hybridization conditions that are substantially above or below the optimum for a given probe can lead to reduced signals. This is most often encountered with probes having unusually high GC or AT content, probes that have a high degree of mismatch with the target, or oligonucleotide probes. Hybridization conditions for these probes may be best determined empirically. See section V.D. Determining Optimum Hybridization Temperature on page 34 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf) for recommendations.
- The probe was degraded. Use radiolabeled probes quickly. The high specific activity probes used in Northerns degrade rapidly due to autoradiolysis, resulting in low signal and/or high background. Make sure non-isotopically labeled probes have not been degraded by nuclease contamination.
- The specific activity of the probe was too low. The specific activity of the probe should be at least 1x10E8 cpm/µg and preferably greater than 1x10E9 cpm/µg. If using end-labeled oligonucleotide probes, switch to a more sensitive probe type such as internally-labeled, longer probes.
- The probe concentration was too low. Calculate cpm (radiolabeled probes) or concentration by A260 (non-isotopic probes) after removal of unincorporated nucleotides. Use 1x10E6 cpm/mL for radiolabeled probes, 1 pM for non-isotopically labeled DNA probes, and 0.1 nM for non-isotopically labeled RNA probes.
- Insufficient exposure. Low copy number RNAs can take >3 days to show up with 32P-labeled probes at -80 degrees C and intensifying screens. With chemiluminescent detection systems, there is often a delay before peak light emission is reached. Low copy number RNAs often take 30 min to 1 hr to show up with the BrightStar system after an initial 2 to 4 hr delay.
- Insufficient target RNA. Load up to 30 µg total RNA. If this is not enough to give a strong signal, poly (A+) RNA should be used (mRNA represents only about 0.5-3% of total RNA). If this is still not enough, switching to a more sensitive technique such as RT-PCR should be considered.
- The message co-migrated with ribosomal RNA. Electrophoresis and/or transfer of target RNA can be hindered by the large amount of ribosomal RNA. Messages that co-migrate with ribosomal RNA may give better signals when poly (A+) selected RNA is used rather than total RNA.
- Inappropriate use of intensifying screens, and exposure temperature. Intensifying screens are only effective if they are incubated with the membrane and film at -70 degrees C. Conversely, if screens are not used, the X-ray film will be much less sensitive at -70 degrees C than at room temperature. When using two intensifying screens, the exposure setup should be as follows: blot - screen - film - screen. The radioactive energy from the blot will go through the adjacent intensifying screen, expose the film and then be reflected back and forth between the two intensifying screens.
- There could have been procedural issues such as:
-Incomplete denaturation of double-stranded probes. Double-stranded probes that are not denatured usually yield little to no hybridization signal. If the probe is only partially denatured, the effective probe concentration will be reduced. Instructions for denaturation of probes can be found in section II.F.3 on page 15 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf).
-Sub-optimal transfer of RNA to membrane. See section IV.C. Problems During Transfer on page 23 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf). -Samples improperly stained with ethidium bromide. Use no more than 10 µg/mL ethidium bromide in Formaldehyde Load Dye.
-Inadequate RNA crosslinking/Over-exposure to UV light. Be sure to follow recommendations for crosslinking of RNA to the membrane. If an uncalibrated hand-held UV source or transilluminator is being used for UV crosslinking, do the pilot experiment for calibrating UV sources. See Ambion Technical Bulletin #169 (https://www.thermofisher.com/us/en/home/brands/invitrogen/ambion.html) for information. Minimize exposure to UV light during UV shadowing.
-Transfer too long/too short. Transfer for 15 min per mm of gel thickness.
-Incompatible membrane. A positively charged nylon membrane, such as Ambion Bright-Star-Plus Membranes, is strongly recommended, particularly for non-isotopic Northerns. Nitrocellulose membranes are not compatible with the NorthernMax Transfer Buffer and should not be used with these kits.
-Failure to follow non-isotopic detection protocols. Follow the manufacturer's recommendations closely. Do not attempt to expose non-isotopically labeled blots to film in a freezer as with radiolabeled blots. Low temperatures will stop the enzymatic reactions and no light will be emitted.

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