Unless otherwise stated, the purity of our products is estimated to be > 98%.

NitroBind pure nitrocellulose, 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. The broad chemical compatibility of PVDF-Plus allows the use of all commonly used stains such as: Amido Black, Colloidal Gold, Coomassie Blue, India Ink and Porceau-S.

Nitro membranes can be autoclaved either way (wet or dry) without concern. On the other hand, AcetatePlus must be autoclaved wet to remain hydrophilic.

Our transfer membranes do not have a specified smooth or rough side. If any variation is observed between the sides of a transfer membrane, it is recommended that the "smooth" side be placed down when performing the hybridization protocol.

Pure nitrocellulose ensures the high binding in both NitroPure and Nitrobind nitrocellulose membrane, approximately 100 μg/cm2.

Yes, alternative transfer systems like vacuum blotting, semi-dry electroblotting, bi-directional transfers, and positive pressure blotting systems can all be used with Osmonics NitroBind and NitroPure membranes. Always remember to follow the manufacturers instructions. The complete protocol for NitroBind and NitroPure Northerns and Southerns is available online at the Osmolabstore.

As a general rule:
DNA oligos
Tight pore size (NLTHY) for <300 bp
Loose pore size (NLTHY) for >300 bp

Protein molecular weight
Tight pore size for <20kD acid, basic
Loose pore size for >20kD acid, basic

The approximate pore size:
Tight = 0.2μm
Loose = 0.45μm

The binding capacity of PVDF Plus is 125μg/cm².

FLOW RATES

   ● PV1HY 1.55 - 7.76 mL/min/cm² @ 10 psi
   ● PV2HY 3.10 - 15.5 mL/min/cm² @ 10 psi
   ● PV4HY 7.76 - 44.3 mL/min/cm² @ 10 psi

Yes, Osmonics manufactures many types of MSI transfer membranes recommended for rehybridization procedures. These include Magna nylon, the MagnaCharge nylon, and the MSI NitroPure, as well as other membranes.

Charged nylon membranes can most frequently be reprobed. MSI MagnaGraph Nylon and MagnaCharge nyylon can be reprobed as many as 10-20 times. MSI Magna nylon, NitroPure and PVDF can be reprobed as many as 5-10 times and NitroBind can be reprobed 2-3 times when special care while handling the membrane is taken.

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 nylon matrix and are not recommended for procedures where reprobing steps are required. The binding capacity of nitrocellulose is less than that of nylon; therefore, the potential number of rehybridizations is fewer.

When performing a western blot with Osmonics PVDF-Plus it is essential to block the membrane. The membrane should be blocked in a solution of 5% BSA in TBS for 1 hour at 37ºC.

A stringent wash would be best when you know your probe is an exact match. The high stringency will wash away all traces of the probe that are not bound perfectly to the target sequence and leave you with low background noise. It is not always possible to obtain such exact probes, so a less stringent wash is desired. Background may be higher, but the probe/target region should remain intact. If you are unsure of your probe/target accuracy it is best to start with less stringent washes and work your way towards higher stringencies. This way you can achieve the ideal results with one transfer.

Osmonic flat sheet PVDF membranes typically run between 70 - 80% porosity.

Poor agitation during pre-hybridization and hybridization steps can lead to insufficient blocking of the entire membrane. Due to the strength of the internal support web used with NitroPure, Magna, MagnaCharge and MagnaGraph transfer membranes, they all withstand higher levels of agitation without tearing or ripping. Incorrect probe concentrations can occur when using dextran sulfate in hybridization or pre-hybridization solutions. Dextran sulfates cause 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 mg/ml of solution. When not using dextran sulfate, maintain the optimum probe concentration of 25-40 mg/ml of solution. Residual agarose of membranes can cause a fuzzy background to appear on blots. Be sure to wash the nylon membrane with the #5 X SSPE at 60°C after the immobilization step. Due to the strength of MSI transfer membranes, they can be more easily washed without tearing or ripping.

PVDF-Plus has a binding capacity of 125 μg/cm2. PVDF-Plus also binds proteins with molecular weights between 5k daltons to 700k daltons.

Osmonics manufactures three types of membranes recommended for rehybridization procedures: MAGNA Nylon, MAGNACHARGE Nylon, and NitroPure, a supported pure nitrocellulose.

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 μm/cm² vs. 400 μm/cm²), the potential number of rehybridizations is fewer.

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 pipette 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.

Yes. UV crosslinking can be used as an additional step to assure attachment of our DNA/RNA to the membrane. By exposing a nylon membrane to a UV source, a covalent bond forms between the membrane and the DNA or RNA which maximizes signal retention after reprobing.

The membrane we recommended for chemiluminescent Southern testing is the MagnaGraph Nylon Transfer Membrane. This membrane is suggested for both single and multiple probes.

Osmonics manufactures the MSI MagnaGraph nylon membrane, the membrane that is most frequently used and recommended by the manufactures of non-radioactive detection systems. The high signal retention and low background of MagnaGraph nylon membranes are frequently cited by these manufactures and are due to the proprietary chemical optimization that makes MagnaGraph transfer membranes easy to block.

Blotchy or incomplete transfers are caused by poor contact between the gel and the membrane. Even after careful smoothing of the membrane up to the gel, incomplete degassing of transfer solutions can cause air pockets. Any trapped gasses can affect the approaching transfer and 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, between the membrane and the chromatography paper. To avoid this problem, roll a pipette down the membrane after it has been applied to the gel and, once again, over the chromatography paper. Do not remove the membrane until the transfer is complete, since this will cause smearing.

Troubleshooting blotting problems begins with the correct gel casting procedure. Skewed, streaked, incomplete or non-uniform transfers can be the result of poorly cast gels. The following recommendations are made for setting up the gel. Gels greater than 4 mm 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 transfer may result. Maintain a gel casting procedure of 55-70°C and be sure the gel particles are completely dissolved. Undissolved agarose particles can result in streaked or skewed bands.

Immediately after 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 that will inhibit transfer of the nucleic acids from the gel. After setting up the blotting assembly, be sure to invert the gel so the underside of the gel is the side that is in contact with the membrane. Allow the transfer solution enough time to breathe, so it may de-gas completely. Incomplete de-gassing of transverse solution results in the evolution of gas after the blotting assembly is set up, which causes air bubbles between the membrane and the gel, which can speed the transfer of nucleic acids.

As with Any membrane overexposure to UV light can decrease your signal intensity. How much UV exposure is recommended?

   ● Wet Membrane ~ 230 seconds
   ● Dry Membrane ~ 23 seconds
   ● These times are based on a 312nm bulb.

Either method is acceptable. There is some safety factor built into the 120 mJ/cm² limit for UV, but if you are used to baking, go ahead and bake! Baking is safer and creates a more reliable, permanent bond. UV crosslinking can degrade the membrane and reduce the strength of the binding and quantity bound.

Keeping a sterile environment is the most important step in ensuring accurate Northerns. The touch of one finger in the wrong place can leave enough RNAse to ruin an entire experiment. It is always a good idea to have separate tools (pipets, pipetters, tips, casting trays, etc.) that are only used for RNA applications. Designating an area of the lab to be used only for RNA work also reduces risk of contamination. When making solutions for your northern always start with sterile ingredients that are only used in your RNA application. After solutions are made they should be again sterilized by filtration or autoclave, if applicable. Remember to use gloves and change them often.

For small volumes
Cameo 25GAS: 25mm Glass Fiber over Acetate Syringe Filter, Sterile

For larger volumes
AcetatePlus VT: Bottle-Top Filtration Units

Over exposure of UV crosslinking will reduce the amount of RNA that will bind to the membrane itself, the membrane will lose sensitivity for binding in general. The antibody will still bind to the RNA but not as much RNA will be on the membrane itself. Decreased signal will be seen on the membrane because there will be less RNA bound to it.

Our NitroPure internally supported 100% pure nitrocellulose transfer membranes are the substitute product for the NitroPlus. This membrane is actually stronger and works better for our customers when compared to the NitroPlus we previously sold.

Osmonics 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.

Osmonics offers several membranes for performing Southerns, they are as follows: Magna, MagnaCharge, MagnaGraph, Nitropure, and Nitrobind.

Here are a few things to consider when dealing with high background:

	●	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, MagnaCharge, MagnaGraph) can be more easily washed without tearing or ripping.
	●	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.

Yes, gels greater than 4mm thick can interfere with free transfer of nucleic acids.

Troubleshooting blotting problems begins with the correct gel casting procedures. Skewed, streaked, incomplete, or nonuniform 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, nonuniform 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.

While there are several ways to decontaminate probe solutions, Osmonics has found that 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 first adding a small volume of the hybridization buffer to the probe, and then filtering it through a Cameo 25AS, low protein binding cellulose acetate sterile syringe filter (Catalog #DDA02025S0). Contaminants in the probe solution will be held back by the 0.2 μm filter, with no probe loss caused by nonspecific binding to the filtration membrane.

Yes, probe length is also a factor contributing to the 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.

Contaminated hybridization solutions are another common source of background problems. Hybridization solutions should be filtered with an Osmonics 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 Cellulose Acetate 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.

We can also offer catalog #DDS92925S0, our Cameo 25SS: 25mm Polyethersulfone sterile syringe filter which is designed for:

	●	High throughputs and rapid flow rates:
	●	Tissue culture media preparation.
	●	Clarification of:

Biological Fluids, Probe Solutions, Protein and Enzyme, Filtration, Hybridization

Osmonics recommends blocking the NitroBind Membrane in PBS containing 5% BSA, Tween 20 or high purity gelatin for 1 hour.

If you are using a radioactive probe:

●

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.

If you are using a non-radioactive probe:

●

Confirm probe removal by blocking with prehybridization solution for 30 minutes before detection, otherwise high background levels will be encountered.

Nitropure is recommended for applications which require Nitrocellulose and extensive handling.
These are:

	●	Southern Transfers
	●	Northern Transfers
	●	Colony/Plaque Lifts
	●	Dot/Slot Blots
	●	Multiple Reprobings

NitroBind is an unsupported Nitrocellulose transfer membrane that can be used for a variety of applications.
These are:

	●	Western Blotting
	●	Immunoblotting
	●	Southern Transfers
	●	Northern Transfers
	●	Dot/Slot Blots

MAGNA nylon, a supported nylon membrane, is optimal for all traditional DNA and RNA transfers.

Applications:

	●	Southerns
	●	Northerns
	●	Dot/Slot Blots
	●	Multiple Reprobings
	●	Radiolabelled Detection Systems
	●	Colony/Plaque Lifts
	●	RNon-Radioactive Detection

Yes we do, the following protocol can be used for Protein Slot-Blotting with NitroBind transfer membrane: Protein Dot/Slot-blotting

If purified, suspend sample in PBS or TBS, or other appropriate buffer. Cell lysates or extracts also may be applied to the membrane or filter for detection of cellular proteins. If using a filtration manifold, dilute sample to 300-500 μl for a concentration of 1-10 μg/spot, then carry out serial dilutions of the sample to determine the optimal concentrations for subsequent binding assays. If not using a manifold, volumes of 2-5 μl should be used.

If using a filtration manifold to apply samples, float membrane in deionized water until completely wet, then soak in PBS or TBS until use. If spotting without a manifold, membrane should be left dry.

Apply sample to membrane directly with a micropipette, or use a filtration manifold. For purified samples, apply 1-10 μg/spot or well. Sample should be diluted in PBS, TBS or other buffer.

Multi-well filtration manifold Place 2 sheets filter paper, prewet in transfer buffer, on the filter support plate of the Manifold device. Place membrane on top of the filter paper and clamp the sample well plate into place. Apply low vacuum (enough to pull buffer through at approximately 1 mL/minute) and wash each well with 500 μL of sample buffer. While the vacuum is applied, add sample to the well and filter through. After sample has been applied to the membrane, wash with 500 μL of sample buffer. For best results, suspend sample in 300-500 μL of buffer. Unclamp and remove the sample well plate, taking care to leave the membrane on the filter paper. Remove the membrane from the Manifold device.

Direct sample application If a Manifold device is not used, apply sample to the membrane placed on top of 2 sheets of dry filter paper in 2-5 μL aliquots. If larger sample volumes are used, allow sample spot to dry prior to subsequent 2-5 μL aliquot addition. Note: If using a filtration manifold, low molecular weight samples should be filtered through slowly to prevent passage through the medium, or applied without vacuum.

Follow blocking and detection procedures as indicated for the NitroBind membrane .

Yes, PVDF-Plus does have a shiny and a dull side. This difference is due to the pores being slightly asymmetrical or "cone shaped". The Shiny side has the smaller opening and the dull side has the larger ones. Either side you choose will offer the same overall pore size. What is important is to be consistent with the side you use.

The gel may be too hard. This agar adhesion is common with hard gels. A possible solution is to reduce the agarose to 0.8-1.5%.

No, transfer membranes are disposable products and rehydration is not recommended after they have dried out. There are no performance guarantees on rehydrated membranes.

With MagnaGraph membranes, the procedure states to not exceed 120 mJ/cm². Going above the 120 mJ/cm² level begins to degrade the membrane. The manufacturers will have a better knowledge of their equipment efficiencies. We do not have any charts to state the binding at various energy levels.

For more information about MagnaGraph membranes, visit our MagnaGraph: Nylon Transfer Membrane page.

Learn about Non-Radioactive Detection probes for hybridizations with MagnaGraph Nylon membranes.

For non-isotopic detection systems, check the Oligonucleotide Probes procedure to be Used in Northern and Southern Blotting For MAGNA, MAGNACHARGE, and MagnaGraph Nylon Membranes.

Also, note the RNA Probes procedure to be Used in Northern and Southern Blotting using MAGNA and MAGNACHARGE and MagnaGraph nylon.

NitroBind is in fact sided, it has both a "shiny" and a "dull" side. The sidedness is due to the pores being asymmetrical. The shiny side has smaller pores than the smooth dull side. As for which side should be used, the answer is just be consistent. You may have to try both sides, but consistently use the one that works best.

The membranes are packaged with the shiny side facing up.

PVDF-Plus is a naturally hydrophobic, unsupported polyvinylidene fluoride transfer membrane designed, manufactured and tested by Osmonics to deliver the highest binding capacity combined with low backgrounds in western transfer, amino acid analysis and protein sequencing applications.

Applications:

	●	Protein Sequencing
	●	Protein Sequencing
	●	Western Transfers
	●	Amino Acid Analysis

Yes. PVDF-Plus does require a wetting agent prior to transfer. We suggest that the membrane be prewet in 5-10 ml of 100% methanol for about 5 seconds.

NitroBind does not contain cellulose acetate, a low binding polymer. This assures the highest binding and sensitivity remains approximately ~100μg/cm² for NitroBind.

Pure nitrocellulose exhibits the highest sensitivity with very low backgrounds in all transfers- especially in protein blotting. Unlike PVDF, nitrocellulose wets out naturally, does not require methanol, and will not turn hydrophobic during the transfer process. Nitrocellulose is very easily blocked and does not need the many blocking steps required with PVDF.

NitroBind and NitroPure must be floated on distilled water and then immersed until thoroughly wet. Then soak the membrane in transfer buffer until use. This is required for both northerns and Southerns.

Formamide allows a lower temperature to be used to melt the DNA. Without Formamide, a higher temperature is needed.

When formamide is used, the hybridization is normally performed at 42°C (108°F) instead of 65°C (149°F).

Formamide-based hybridization solutions are a frequent source of background noise, and the formamide must be freshly made and deionized. The solution should also be filtered with our Cameo 25GAS: 25mm Glass Fiber over Acetate Sterile Syringe Filter, DGA02025S0 or our Cameo 25GSS: 25mm Glass Fiber Over Polyethersulfone Sterile Syringe Filter, DGS02025S0 to help eliminate background.

The membrane requires baking or UV crosslinking. Osmonics recommends baking procedure for stronger binding to the membrane. This is recommended for customers that may not be famaliar with the UV apparatus.

UV can be used but may not be as reliable as baking and over-exposure will degrade the membrane and lower the signal. For optimum UV parameters variation may occur depending on brand, membrane type and wetness, size and type of nucleic acid, transfer buffer used, age and condition of the UV bulbs for some examples. Many UV end-user prefer the UV equipment, requiring only seconds versus 2 hours for baking, because the variables have been worked out and are established. It is easier to use.

One of our customers was wondering if drying E. coli on Magna Lift nylon transfer membranes prior to denaturation effects the colony hybridization. Further, they were wondering if after drying the membranes, would treating with SDS before denaturation effect the hybridization? The answer is ... no, this will work! The hybridization will not be adversely affected. This is the procedure they used.

Procedure

	1.	performed colony lifts
	2.	air dried nylon membranes with E. coli
	3.	incubated 3 minutes on 3MM paper saturated with 10% SDS
	4.	incubated 5 minutes on 3MM with 0.5M NaOH, 1.5 M NaCl
	5.	incubated 5 minutes on 3MM with 1.5 M NaCl, 0.5M Tris pH 7.5
	6.	incubated 5 minutes on 3MM with 2X SSC
	7.	UV crosslinked without drying
	8.	prehybridized and hybridized as usual

Magna Nylon is recommended for DIG labeling hybridizations. Remember to always check the recommendations of the kit manufacturer first.

Osmonics recommends using NitroBind unsupported Nitrocellulose membranes for protein applications in which non-radioactive probes are to be used. The NitroBind will yield lower background than a PVDF membrane with non-radioactive probes.