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Millipore Technical Publications


AN- WWW1

Blotting Optimization --Nylon Blotting Transfer Membranes for Nucleic Acid Applications

Lit No:WWW1
Year:2001



Blotting is the process of transferring electrophoretically separated biomolecules (e.g., nucleic acids or proteins) from a gel (agarose or polyacrylamide) onto a membrane substrate. Membranes have a large internal surface area and are made from highly adsorptive polymers. Both of these attributes make them an ideal substrate for the binding and retention of biomolecules. As compared to the gel matrices from which the biomolecules are transferred, membranes are easier to handle and can be manipulated more effectively in subsequent detection methods.

Biomolecules can be transferred from the gel to the membrane using capillary force, vacuum, or electrical field. Colonies (bacteria) or plaques (viruses) are grown on agar plates and can be transferred by laying the membrane on the plate. Although the basic techniques for preparing and analyzing blots are more than two decades old, recent improvements in the understanding of blotting techniques have given way to broader utilization of blotting membranes in the analysis of nucleic acids.


Immobilon Nylon Transfer Membranes

Life scientists have long relied on different types of membranes for nucleic acid hybridization techniques. Nitrocellulose membranes are electrostatic, somewhat brittle and difficult to reprobe. Nylon membranes cast from nylon 6,6 polymer(1,2) have superior handling characteristics relative to nitrocellulose membranes and are better able to withstand the chemical conditions for stripping DNA or RNA probes. The use of nylon membranes in nucleic acid blotting applications has improved the performance and capabilities of hybridization-based assays.

Millipore offers two hydrophilic nylon membranes: Immobilon-Ny+,which is positively charged, and Immobilon-Ny, which is uncharged. Both membranes are made from web-supported nylon 6,6 with a 0.45 µm pore size. The open pore structure permits maximum accessibility of target sequences to the probe and allows efficient removal of unhybridized probe, thereby reducing background. There are differences in the binding mechanism of the two membranes. The charged Immobilon-Ny+ binds primarily by ionic interactions (and covalently after UV fixation). The uncharged Immobilon-Ny binds primarily by electrostatic interaction (and by covalent binding after UV fixation).

Millipore ’s nylon membranes have been shown to be highly useful substrates for Southern blotting, northern blotting and colony lifts.However, there is considerable variability in the performance of different nylon membranes. The uncharged Immobilon-Ny nylon membrane can be used with traditional protocols to yield equivalent or better results (e.g., reduced background interference in chemifluorescence and northern blots). In our laboratories, charged Immobilon-Ny+ membrane has greater sensitivity than Immobilon-Ny when used with optimized protocols.

Key Factors for Successful Southern Blotting

Southern blotting of DNA to a microporous membrane(3) was a breakthrough technique in molecular biology. In blotting, the most important performance criterion is sensitivity of detection. To maximize detection sensitivity, four factors are critical:
1. Maximum transfer of biomolecules from the gel.
2. Maximum binding and retention of the transferred biomolecules on the membrane throughout subsequent analyses.*
3. Maximum accessibility of all membrane-bound biomolecules to the probe.
4. Maximum detection sensitivity and minimum background.

*For nucleic acid blotting applications,the DNA and RNA binding capacity of the membrane are normally not limiting. The quantities of DNA and RNA used in routine experimentation are not high enough to saturate the membrane.

1 . Transfer from the Gel

Maximum sensitivity on Immobilon-Ny and Ny+ membranes is achieved using capillary transfer in 20 x SSC buffer followed by fixation of the DNA by UV cross-linking.(4) The DNA should be denatured before being filtered or transferred onto the membrane, and standard transfer protocols include a step for denaturation in alkali prior to transfer. Transfer under alkaline conditions(5) has been reported to fix DNA to the nylon surface without the need for UV exposure.Under alkaline conditions, DNA transferred to Immobilon-Ny+ typically shows a lower and inconsistent hybridization signal when compared to the 20 x SSC control with UV fixation. This process may not allow for denaturation of the DNA prior to its immobilization on the membrane, thereby limiting its accessibility during hybridization. Alkaline transfer is not recommended for maximal hybridization signal.


Figure 1. Comparison of DNA Transfer Methods. .

Lambda DNA, digested with Hind III, was resolved on agarose gels and transferred to Immobilon-Ny+ using 20 x SSC transfer or alkaline transfer. Half of each blot was cross-linked by exposure to UV light prior to hybridization. Images were generated by phosphor imaging...


Tips and Techniques: To maximize elution from the gel, maintain a low agarose concentration and depurinate the DNA. To avoid premature collapse of the gel, do not use a heavy weight during the transfer.


2 . Binding and Retention

Fixation methods are commonly used after blotting to bind the nucleic acids to the membrane more effectively.It is very important that nucleic acids are fixed to the membrane at optimal conditions and that the blot is analyzed using conditions that produce the highest signal-to-noise ratio. Fixation procedures vary depending on the blotting membrane that is chosen. Nucleic acids can be fixed to nitrocellulose membrane by baking at 80 °C under vacuum. UV cross-linking is the preferred method for fixing nucleic acids to nylon membranes. Short wavelengths (254 nm) of UV light will cross-link the DNA by covalent interaction with the primary amines on the membrane surface(6) resulting in high retention.

Tips and Techniques The blot must be dried completely prior to UV irradiation because water molecules in the pore structure quench the UV energy.


3 . Maximum Accessibility

The accessibility of the probe to the target during hybridization and the physical retention of the target on the membrane surface are two important parameters that must be balanced to achieve optimal hybridization. Maximum hybridization signal is achieved at UV exposures of 5000 µJoules/cm2 for blotted DNA and 1700 to 5000 µJoules/cm2 for filtered DNA at 254 nm for Southern blots. At higher energy levels, significant structural cross-linking of the target DNA improves retention but sterically hinders hybridization. In contrast, RNA requires a higher UV energy to achieve optimal hybridization efficiency: 20,000 µJoules/cm2for DNA probes and 40,000 µJoules/cm2 for RNA probes.This is most likely related to the presence of uracil instead of thymidine in the RNA structure and the consequential reduction of covalent bonds.


4 . Detection and Background

To detect the bands on the membrane, a probe specific to the target band is used. Probes, such as DNA or RNA fragments that have a sequence complementary to the target sequence, are hybridized to the target band on the membrane. The probe may be labeled with radioisotope before hybridization and visualized by phosphor imaging or exposure to X-ray film after post-hybridization washes. Non-radioisotopic detection methods have also been described that utilize chemiluminescence or chemifluorescence. In these instances, the probe may be directly labeled with hapten molecules to facilitate subsequent binding of a fluorescently-labeled secondary probe molecule and then visualized by detecting the secondary probe molecule.

Reprobing, the multiple probing of transferred nucleic acids, is now commonly performed. After the first target band has been detected, the probe is removed (stripped) from the membrane and a second probe is hybridized (reprobing to detect the next target band. The reprobing continues until all bands of interest have been detected. Low abundance targets should be probed first and high abundance targets should be probed last. The repetitive steps of stripping and reprobing reduce the time, labor, and cost of blot preparation (e.g.,gel electrophoresis, pretreatment of gels and transfer to membranes).

Tips and Techniques

  • The blot must be dried completely prior to UV irradiation because water molecules in the pore structure quench the UV energy.
  • Immobilon-Ny+ and Immobilon-Ny should not be dried during the hybridization, washing or detection steps if the membrane will be reprobed. Drying causes the probe to bind irreversibly to the membrane.


Detection Methodologies

Non-radioactive detection methodologies provide several advantages over radioactive methods, including fewer limitations on waste handling and extended stability of chemicals and labeled probes. However, low detection sensitivity and background issues are sometimes encountered with commonly available non-radioactive detection systems.Obtaining good results with non-radioactive detection systems depends in large part on the performance of the membrane and the protocol utilized.


Figure 2. Hybridization on Immobilon-Ny+ after Exposure to Low Level UV Energy.

DNA was applied to Immobilon-Ny+ by blotting from an agarose gel. After exposure to UV light at 254 nm, the blots were hybridized and the signals were quantified. The hybridization signals were normalized to the untreated control for that set. Each data point represents the mean of two values..



A series of experiments with non-radioactive detection using chemiluminescence has demonstrated that Immobilon-Ny+ shows better performance than two competitive, positively charged nylon membranes. Maximum detection sensitivity on Immobilon-Ny+ using chemiluminescence is achieved with fixation of DNA by UV cross-linking at optimal conditions followed by hybridization in modified Church hybridization solution (0.5 M sodium-phosphate, pH 7.1, 2 mM EDTA, 7%(w/v) SDS, 0.1%(w/v) sodium pyrophosphate).The optimum range for UV cross-linking DNA onto Immobilon-Ny+ is between 2,500 and 10,000 µJoules/cm2 This optimal energy is consistent with the optimum energy for radioactive probes.Immobilon-Ny+ showed more than two-fold higher sensitivity with 10 ng of lambda Hind III DNA in a two-hour film exposure.(7)

For successful results in non-radioactive detection systems, the following considerations should be taken into account when optimizing the protocol:
  1. The optimal energy for UV cross-linking is 5,000 µJoules/cm2 at 254 nm.Higher UV energies and other wavelengths are not recommended for maximum sensitivity.
  2. The probe concentration should be approximately 10 ng/mL in the hybridization buffer. Lower concentrations reduce sensitivity. At concentrations >20 ng/mL, the background rapidly increases and can not be reduced by extending the washing times increasing the number of washes. The concentration of freshly labeled probe must be determined by analyzing control DNA in a dot blot assay.
  3. The modified Church hybridization solution improves detection sensitivity by allowing extended exposure times. The solution does not contain blocking agents derived from milk, which may interfere with detection systems employing bioti /avidin recognition.
  4. Solutions should be filtered through a 0.22 or 0.45 µm filter prior to use, in order to remove particles that may contribute to background.


Stripping Protocols

Nylon membranes are more durable than nitrocellulose, making nylon ideal for applications where numerous rounds of stripping and reprobing are required.During hybridization and post-hybridization washes, DNA or RNA may be rinsed off the membrane. Fixation with optimal UV energy is highly recommended. Four stripping protocols were evaluated on Immobilon-Ny+ and a competitive membrane(8). All protocols left <3%of the initial 32 P probe on the Immobilon-Ny+ blots when the hybridization probe was used at a concentration of 10 ng/mL. Immobilon-Ny+ had less residual probe signal than the competitive membrane. The signals on Immobilon-Ny+ were also 4.5 to 5.5-fold higher than the competitive membrane. In addition, Immobilon Ny+ membrane was stripped more efficiently.(8)



Figure 3. Comparison of the Four Stripping Methods on Immobilon-NY+

. Note: The values are % of initial probe.


A variety of stripping protocols works with Immobilon-Ny+ and Immobilon-Ny membranes. Either the Denaturation/Neutralization or the 0.5%SDS stripping method is recommended for Immobilon-NY+. The 50% formamide method is not recommended. DNA probes can be stripped from the membrane by incubation of the blot in 0.4 N NaOH at 45 °C for 30 minutes followed by neutralization for 15 minutes at room temperature in 0.1 x SSC, 0.1%(w/v) SDS,0.2 M Tris-HCl, pH 7.5. Alternatively, the probe can be stripped by placing the blot in boiling 0.5% (w/v) SDS and allowing the solution to cool to room temperature. Probes on northern blots can be stripped by heating blots twice in boiled 0.1%SDS with gentle agitation for 15 minutes. Signal intensity decreases dramatically upon reprobing if 50 mM NaOH is used.


Reprobing

Immobilon-Ny+ membrane was compared to two competitive membranes following transfer from an agarose gel in 20 x SSC and optimal UV fixation for each membrane. Immobilon-Ny+ membrane exhibited two-fold higher signal than competitors A and B when detected with a 32P-labeled probe. After reprobing, Immobilon-Ny+ maintained a higher level of signal compared with the competitor membranes through 12 cycles of reprobing (see page 29). In reprobing analysis, a background signal can be generated from the nonspecific binding of a probe or residual signal left from an earlier round of hybridization. Immobilon-Ny+ membrane,when used with the recommended modified Church hybridization solution,showed a consistently lower background signal in chemiluminescent detection systems.(7)



Figure 4. Quantification of Cyclophilin Signal after First Probing.

Mouse liver total RNA (1 µg) was resolved in a formaldehyde agarose gel and transferred to Immobilon-Ny+ and competitor's A by capillary blotting. The blots were dried completely then fixed with either baking (80 °C for 1 hr) or UV cross-linking (UV 20, UV 40, and UV 60: 20,000, 40,000, and 60,000 µJoules/cm2 at 254 nm, respectively). The blots were hybridized using either DNA or RNA of cyclophilin probe and exposed to a phosphor screen for 6 hr. The bands then were quantified. The bars labeled UV20/40 in the graph refer to UV cross-linking with the optimal UV energy for either DNA (20,000 µJoules/cm2 ) or RNA (40,000 µJoules/cm 2 ) probes. Ny+: Immobilon-Ny+. A: competitor's positively charged nylon.



Northern Blotting

Millipore has systematically determined the conditions that produce the best results on northern blots using Immobilon-Ny+ membrane. Experimental data were quantified by phosphor imaging analysis.RNA fixation by UV cross-linking enhances signal relative to the baking method. The optimal energy recommended at 254 nm is 20,000 µJoules/cm2 for DNA probes and 40,000 µJoules/cm2 for RNA probes. Modified Church hybridization solution works well for northern blots. Formamide may be added to the hybridization solution at concentration of 33 to 50%(v/v)to prevent cross-hybridization of an RNA probe to ribosomal RNA.

The stripping method using hot 0.1%(w/v)SDS is recommended for northern blots. Do not use sodium hydroxide to strip Northern blots. Signal intensity decreases dramatically upon the first re-probing after the blot is stripped with 50 mM NaOH, most likely due to alkali-induced hydrolysis of theRNA. For reprobing on northern blots, hybridization with DNA probes at 68 °C produces better results than RNA probes in formamide-based buffers. Also, probes should be used in an order so that low abundance targets are analyzed before high abundance targets.

The length of time that a blot can be held in storage depends on the quality of the blot (free from RNase) and the sensitivity required (abundance of target RNA and how much sample RNA was loaded).Depending on these conditions, the blots may be stable at 4 °C for a month with the band of interest still detectable without any signal decrease. Place the dried northern blots between clean Whatman® 3MM filter papers in a clean plastic bag and store them at 4 °C in the dark. Alternatively, the dried blots may be stored at –20 °C.Long-term storage is not recommended for wet blots.

Colony Lifts

It is unnecessary to wet and autoclave the Immobilon-Ny+ and Immobilon-Ny membranes prior to a colony lift unless there is a concern about carrying over viable microbes on the membrane.The membrane can be placed dry onto the plate for the lift.The plates should be chilled at 4 °C for about 30 minutes prior to the lift to prevent the medium from lifting with the membrane.

If sterilization of the membrane is required, wet the membrane by laying it on top of ultrapure water from a Milli-Q® system and letting the water slowly absorb through the membrane. This will prevent airlock,which could occur if the membrane is quickly immersed in the water. Sandwich the wet membranes between dry sheets of Whatman 3MM filter paper. Wrap the pile of membranes in aluminum foil. Autoclave at 15 lb./sq. in (1.05 kg/cm2 )at 121/°C on a liquid cycle (slow exhaust).The optimal UV energy for maximum UV cross-linking is 60,000 µJoules/cm2 for colonies that are less than 1 to 2 mm in diameter and 120,000 µJoules/cm2 for colonies that are 3 to 5 mm in diameter.

Effect of Water on Blotting

Water quality has an effect on the overall outcome of the results.Ultrapure water from a Milli-Q system or equivalent water with >18.2 Megohm-cm resistivity should be used to prepare all reagents. Water systems should be well maintained to avoid the build-up of organic contaminants, pyrogens, and nucleases.

Some facts to keep in mind:

  • ucleases will digest probes.
  • articulates can lead to high backgrounds by serving as sites for non-specific binding of probe molecules.
  • eavy metal ions can bind to DNA and RNA,altering their structure and inhibiting hybridization.
  • yrogens will inhibit horseradish peroxidase,which is used as the enzyme in many non-radioactive kits.
  • ariable water quality can cause variable results that are independent of the membrane.

Although the techniques of Southern blotting, northern blotting, and colony and plaque lifts are more than 20 years old, they have not been supplanted by more recently developed techniques for nucleic acid analysis. Instead, they have become complementary to other molecular biology techniques, such as site-directed mutagenesis, gene expression, and sequencing. Continued characterization of membranes, blotting processes, and detection technologies have given rise to major improvements in the utility of these methods. Today, this knowledge is being used to develop products and protocols for high-throughput screening procedures where blotting membranes have been found to be the preferred substrate. For the foreseeable future, blotting technology will continue to make a significant contribution to the analysis of nucleic acids.

References

1.Gershoni, J.M.Transfer of macromolecules from a chromatographic substrate to an immobilizing matrix.US Patent #4,001,828 issued July 22,1986, assigned to Yale University, New Haven,CT.
2.Marinaccio, P.J.and R.A.Knight.Process for producing microporous films and products.US Patent #3,876,738 issued April 8,1975, assigned to AMF, White Plains, NY.
3.Southern, E.M.(1975).Detection of specific sequences among DNA fragments separated by gel electrophoresis. J.Mol.Biol.98, 503-517
4.Millipore Corporation Optimization of DNA fixation to Immobilon-Ny+using ultraviolet light. Technical Note No.TN054,Bedford, MA.
5.Chomczynski, P., (1992). One-hour downward alkaline capillary transfer for blotting of DNA and RNA.Anal.Biochem.201, 134-139
6.Church, G.M. and W.Gilbert.(1984).Genomic sequencing. Proc.Natl.Acad.Sci.USA 81, 1991-1995.
7.Millipore Corporation Chemiluminescent detection of blotted DNA on Immobilon-Ny+. Technical Note No.TN071.Bedford, MA.
8.Millipore Corporation. Comparison of four stripping protocols for DNA probes on Immobilon-Ny+ Technical Note No.TN056.Bedford, MA.