Selecting a Transfer Membrane

Selecting a Membrane

Selecting the appropriate membrane is critical to the success of a nucleic acid or protein transfer procedure. GE Osmonics manufactures many types of membranes for hybridization technology, each exhibiting different performance characteristics which can directly affect the outcome of a specific technique. Below are some of the more frequently performed procedures and features of hybridization membranes.

Rehybridizations

GE Osmonics manufactures membranes recommended for rehybridization procedures: Magna Nylon, MagnaProbe Nylon andNitroPure, a supported pure nitrocellulose.

MagnaProbe Nylon membranes can be most frequently reprobed. On nylon membranes, the number of reprobing steps is a function of the amount of hydrolysis to which the membrane is exposed during the protocol, and the additive effects of hot water, sodium hydroxide and an acidic environment. Sodium hydroxide solutions deteriorate the nylon matrix and are not recommended in procedures where reprobing steps are required.

The polyester support web used in manufacturing NitroPure allows the membrane to be reprobed several times. Because the binding capacity of nitrocellulose is less than that of nylon (100 µg/cm² vs. 400 µg/cm²), the potential number of rehybridizations is fewer. Click here for more details.

Non-Radioactive Probes

MagnaGraph Nylon membrane is frequently recommended by companies that manufacture non-radioactive detection systems. The high signal retention and low backgrounds of MagnaGraph nylon are frequently cited, and are due to the proprietary chemical optimization of the membrane that makes MagnaGraph very easy to block. MagnaProbe Nylon may be used particularily where multiple probing is involved.

UV Crosslinking

For covalent binding of nucleic acids to a transfer membrane, GE Osmonics membranes can be UV Crosslinked by following the manufacturer's instructions. It is particularly recommended when working with short fragments, small samples, or low numbers of base pairs, because of the improved resolution this technique offers.

Protein Blotting

NitroBind pure nitrocellulose and PVDF-Plus membranes are recommended for use in protein blotting. Nitrocellulose membranes are able to be more thoroughly blocked, reducing the high background potential associated with protein blotting. PVDF membranes are more resistant to the harsh chemicals used in Edman degradation.

Low Signal Binding Procedures

The high binding capacity of MAGNACHARGE nylon makes these membranes highly recommended for use with short fragments with a very low number of base pairs, (as low as 72 bp) or with very small amounts of sample.

Alkaline Blotting

For more rapid transfers, an alkaline blotting procedure can be used with MagnaProbe or MagnaCharge membranes. Alkaline blotting is not recommended when reprobing is required. Click here for more details.

Staining Procedures

NitroPure, NitroBind and PVDF-Plus membranes are recommended for procedures that require a staining step with India Ink, Coomassie Blue, Colloidal Gold, or any other commonly used stain. Nylon membranes irreversibly bind many stains.

Reducing Backgrounds

There are many sources of background problems, or low signal-to-noise ratios. Some of the most common include: contaminated probes, contaminated hybridization solutions, and incorrectly chosen stringency levels. Nonfat milk should not be used as a blocking agent as it may increase nonspecific binding. Nylon membranes have a much higher binding capacity than nitrocellulose membranes, and a greater potential for backgrounds. GE Osmonics membranes are all manufactured by strict quality control procedures, ensuring a uniform membrane with consistently low backgrounds. 

Troubleshooting Common Blotting Problems

Many blotting problems can be eliminated by observing the following recommendations.

Blotchy or incomplete transfers are caused by poor contact between the gel and the membrane. Even after careful smoothing of the membrane to the gel, incomplete degassing of transfer solutions can cause air pockets to form. Evolving gas from Tris or, in the case of protein transfers, methanol, can disrupt the tight contact necessary between the membrane and the gel for successful transfers.

Smeared or skewed bands are often caused by uneven contact between the gel and the membrane, or the membrane and the chromatography paper. To avoid this problem, roll a pipet down the membrane after it has been applied to the gel, and once again over the chromatography paper after it has been applied to the membrane. Do not move the membrane until the transfer is complete, as this will cause smearing.

Troubleshooting Guide and Application Tips

Problems and Solutions

Unsuccesful Rehybridizations

My membrane is deteriorating during the rehybridization procedure?

If so, what type of membrane are you using? GE Osmonics manufactures three types of membranes recommended for rehybridization procedures: Magna Nylon, MagnaProbe Nylon, and NitroPure, a supported pure nitrocellulose.

Magna Nylon membranes have largely replaced nitrocellulose because they are more resilient during applications requiring multiple reprobes. Nitropure (a supported nitrocellulose) was developed for the same reason. The polyester support web used in manufacturing NitroPure allows the membrane to be reprobed several times. MagnaProbe Nylon is similar in resiliency to Magna Nylon.

My application demands an extensive number of reprobes and I’m losing signal?

If so, what type of membrane are you using? Because the binding capacity of nitrocellulose is less than that of nylon (100 µm/cm² vs. 400 µm/cm²), the potential number of rehybridizations is fewer as compared to nylon membranes. Likewise, the binding capacity of charged nylon (MagnaProbe) membranes is generally greater than standard nylon, and subsequently charged nylon may have advantages during multiple reprobes. The number of reprobing steps is a function of the amount of hydrolysis to which the membrane is exposed during the protocol, and the additive effects of hot water, sodium hydroxide and an acidic environment.

My probe is not stripping from the membrane, how should I change my procedure?

Did you let the membrane dry after the initial probe was applied? Drying causes irreversible binding of DNA to microporous membranes. If this has occurred, look through the helpful tips listed below.

My probe won’t strip from the membrane, how can I rescue this blot?

Try preparing a new probe and using a different detection protocol. For example, if you prepared a biotinylated probe and detected with a streptavidin conjugate, omit the biotin-streptavidin step during rehybridization by using a directly conjugated probe, such as an alkaline phosphatase conjugated probe. If you used a radioactive probe, use a chemiluminescent system to detect after the next hybridization (or vica versa). If you have enough time and are using radioactive probes (e.g., pgs 106-107), simply let your first probe decay before the second round of hybridization.

Signal Problems

The nucleic acid did not transfer completely to the membrane, what should I do?

Blotchy or incomplete transfers are caused by poor contact between the gel and the membrane. Even after careful smoothing of the membrane to the gel, incomplete degassing of transfer solutions can cause air pockets to form. Evolving gas from Tris or, in the case of protein transfers, methanol, can disrupt the tight contact necessary between the membrane and the gel for successful transfers.

My Signal is low, what are the common reasons for this?

When you have low signal, it is best to check your reagents by performing extra controls. The most common reason for poor signal is a bad probe. Prepare a new probe and perform a dot blot comparing the old and new probes. Do you see a difference between the probes? Even nonradioactive probes can deteriorate during storage. Is the signal weak for the new probe as well? Then your detection enzymes may be bad or the reagents used to prepare the probe are bad. You might also blot a small amount of unlabeled complementary DNA and hybridize to the new probe. Are you seeing signal from the blotted probe but not the hybridized DNA? If so there could be a problem with your hybridization protocol, such as the wash temperature or your buffers. If you’re using nonradioactive detection methods, test your enzymes and substrates as well.

Background Problems

I switched to a non-radioactive detections system, and now I have high background, what should I do?

MagnaGraph Nylon membrane is frequently recommended by companies that manufacture non-radioactive detection systems. The high signal retention and low backgrounds of MagnaGraph nylon are frequently cited, and are due to the proprietary chemical optimization of the membrane that makes MagnaGraph very easy to block. MagnaProbe Nylon may be used particularily where multiple probing is involved. Additionally, GE Osmonics positively charged MagnaProbe membrane shows superior results as compared to competitor membranes when non-radioactive detection is used.

Everything was working fine and now suddenly I have high backgrounds, Why?

Did you make up a new probe? If so, was there adequate separation of the unincorporated label from the incorporated? Are you using old solutions? There may be contamination. Usually in these cases it is best to prepare new solutions, new probes and use new reagents. This is often the fastest way to get your system working again.

Miscellaneous

My membrane changed color during my blotting procedure, should I be concerned?

No. Slight color changes in GE Osmonics new positively charged membranes are expected and have no effect on results. These color changes will vary according to the blotting procedure used and the pH of solutions. GE Osmonics uses this color change to ensure quality during the manufacturing procedure.

High Backgrounds

Poor agitation during prehybridization and hybridization steps can lead to insufficient blocking of the entire membrane. Due to the strength of the internal support web, NitroPure, Magna, MagnaProbe, MagnaCharge and MagnaGraph can withstand higher levels of agitation without tearing or ripping. Incorrect probe concentration can occur when using dextran sulfate in hybridization or prehybridization solutions. Dextran sulfate causes the effective concentration of the probe to increase because it excludes the probe from the volume of solution the dextran sulfate polymer occupies. When using dextran sulfate, lower the probe solutions to less than 10 ng/ml of the solution. When not using dextran sulfate, maintain the optimum probe concentration at 25-40 ng/ml of solution.

Residual agarose on membranes can cause a fuzzy background to appear on blots. Be sure to wash nylon membranes with 5 x SSPE at 60°C, after the immobilization step. Due to the strength of the membrane, supported membranes (NitroPure, Magna, MagnaProbe, MagnaCharge and MagnaGraph) can be more easily washed without tearing or ripping.

Troubleshooting Gel Casting Procedures

Troubleshooting blotting problems begins with the correct gel casting procedures. Skewed, streaked, incomplete, or non-uniform transfers can be the results of poorly cast gels. The following recommendations are made for setting up the gel. Gels greater than 4mm thick can interfere with the free transfer of nucleic acids.

Be sure that the gel tray is level before casting the gel. If the surface is not level, non-uniform transfers may result. Maintain a gel casting temperature of 55-70°C degrees, and be sure that the gel particles are completely dissolved. Undissolved agarose particles can result in streaked or skewed bands.

Immediately after gel casting and solidification, submerge the gel slab in electrophoresis buffer. This will prevent the formation of an impermeable "skin" over the surface of the gel which can inhibit transfer of nucleic acids from the gel.

After setting up the blotting assembly, be sure to:

• Invert the gel so that the underside of the gel is the side in contact with the membrane.

• Allow the transfer solutions enough time to "breathe," so that they may degas completely. Incompletely degassed transfer solutions evolve gas after the blotting assembly is set up, and can cause air bubbles between the membrane and gel that can impede the transfer of nucleic acids.

Probe Related Background Problems

While there are several ways to decontaminate probe solutions, the following methods are two of the most efficient. The second method can be rapidly performed with minimum effort.

Method 1:

Phenol/Chloroform extract the probe to remove unincorporated nucleotides, proteins, and other contaminants.

Method 2:

Clean the probe by adding a small volume of the hybridization buffer to the probe and filtering it through a Cameo 25AS low protein binding cellulose acetate syringe filter (Catalog #DDA02025S0). Contaminants in the probe solution will be held back by the 0.2µm filter with no probe loss caused by nonspecific binding to the filtration membrane.

Probe length is also a factor contributing to background levels seen on transfer membranes. Between 250-800 base pairs is the recommended optimum length of a probe; probe lengths smaller or larger than this can lead to a low signal-to-noise ratio. Probes smaller than 250 base pairs often bind poorly and may require less stringent hybridization and wash procedures. Probes larger than 800 base pairs may contain a wider variety of size classes, which can lead to extraneous binding to the transfer membrane.

Hybridization Solution Related Background Problems

Contaminated hybridization solutions are another common source of background problems. Hybridization solutions should be filtered with a pure cellulose acetate Cameo 25AS syringe filter (Catalog #DDA02025S0), to remove contaminants.

Additionally, all solutions and buffers should be made fresh before each transfer with sterile, double-distilled, deionized water, and very high grade reagents. After fresh buffers are made, they should be filtered with a Cameo 25AS syringe filter (Catalog #DDA02025S0) to ensure that no contaminants remain in the solution. Formamide-based hybridization solutions are a frequent source of background noise, and the formamide must be freshly made and deionized.

DNA Dependent Background

This type of background is caused by nonspecific annealing of the probe to the bound DNA or RNA, and can be eliminated by adjusting the stringency of both the hybridization and wash steps, and by the addition of heterologous DNA or RNA to the hybridization solutions. During the hybridization and prehybridization steps, a concentration of 0.1-1% SDS is recommended for nylon membranes. Because the final wash step after the hybridization is the most important,

GE Osmonics recommends:
Northerns: 0.1 x SSPE, 0.1-0.5% SDS, for 30 minutes at 65°C.
Southerns: 0.1 x SSPE, 0.5-1% SDS, for 30 minutes at 65°C.

Optimized Blocking Solutions

A concentration of 5-7 x Denhardt’s solution is recommended for use with nylon membranes. Exceeding this level can lead to quenching of the signal.

Backgrounds Associated with Reprobing

A follow-up autoradiograph after probe removal is strongly recommended to determine if the probe has been fully stripped. Otherwise, backgrounds can appear in blots that have not been fully erased.