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Detection Membranes

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Pall Life Sciences offers a broad range of polymeric membranes for use with common protein and nucleic acid transfer and detection procedures.

 

Please see specifications for a description of these membranes, tips for selecting the best product for a particular application, and basic procedures for use. Specific applications may require modifications to these procedures


Characterization:

Membranes from Pall Life Sciences

Biodyne® Nylon MembranesBiodyne Nylon 6,6 membranes provide excellent resolution, high sensitivity, and low background. Biodyne membranes are inherently hydrophilic and do not require prewetting. These membranes are considered the “gold standard” for critical molecular biology applications such as forensic detection, and are recommended for all nucleic acid detection procedures as well as protein dot ELISAs. Biodyne membranes do not shrink, crack, or tear when subjected to multiple cycles of hybridization, stripping, and reprobing. Four different surface chemistries are available to address a variety of applications. (See Transfer and Affinity Membrane Selection Guide below for more information.)

Biodyne A membrane is an uncharged Nylon 6,6 membrane. It has a high binding capacity for biomolecules and offers high sensitivity and nucleic acid retention. It is especially well suited for use with chemiluminescence or other detection systems where background is problematic.

Biodyne B membrane has pore surfaces populated by a high density of quaternary ammonium groups, making it strongly cationic. This positive charge is maintained over a broad pH range and promotes strong ionic binding of nucleic acids, making it ideal for transfer and detection of nucleic acids. Biodyne B membrane will produce the highest possible sensitivity with radioactive probes.

Biodyne Plus membrane, like Biodyne B membrane, contains pore surfaces populated by a high density of quaternary ammonium groups. Biodyne Plus membrane is recommended for detection systems that produce high background on Biodyne B membrane. Biodyne Plus membrane often has better sensitivity in these systems than Biodyne A membrane.

Biodyne C membrane is negatively charged with pore surfaces populated by a high density of carboxyl groups. The membrane can be derivatized by coupling reactions with these groups. It is ideal for reverse dot blots and affinity purification.

BioTrace™ NT Membrane

BioTrace NT membrane is composed of pure, unsupported nitrocellulose. Its high tensile strength makes it less likely to tear or crack than other brands of nitrocellulose. The membrane has a high binding capacity for both proteins and nucleic acids and is ideal for colony/plaque lifts and protein transfers.

Pall PVDF Membranes

PVDF (polyvinylidene fluoride) can be formed into hydrophobic microporous membranes. These membranes will exhibit very high binding of both proteins and nucleic acids, but are most useful for immobilization and detection of proteins.

BioTrace PVDF membrane performs especially well with chemiluminescent and colorimetric detection systems. It is highly resistant to organic solvents and aggressive aqueous solutions. BioTrace PVDF membrane is recommended for all protein transfers.

FluoroTrans® membranes offer the highest available binding of proteins (through hydrophobic interactions). FluoroTrans membrane is recommended for N terminal protein sequencing and Western transfers with immunodetection.

FluoroTrans W membrane is optimized for Western transfer applications. This membrane allows for sensitive protein detection with low background and very low protein burn-through. FluoroTrans W membrane is compatible with all total protein stains and immunodetection methods.

UltraBind™ Membrane

UltraBind (modified polyethersulfone) membrane contains active aldehyde groups and does not require special pretreatment prior to use. Proteins and peptides can be covalently attached via reactions between their free amino groups and the aldehyde groups on the membrane. It is ideal for solid phase ELISAs.

 

Transfer and Affinity Membrane Selection Guide

Pall Life Sciences offers membranes for use in transfer and immobilization procedures. These membranes can be used for nucleic acid and protein applications and are compatible with radioactive and nonradioactive detection systems. 

Product Biodyne A Membrane Biodyne B Membrane
Description Amphoteric Nylon 6.6 Positively-Charged Nylon6,6
Recommended for Colony/plaque lifts
DNA and RNA transfers
DNA fingerprinting (non-
radioactive)
ELISA
DNA and RNA transfers
Multiple reprobes
DNA fingerprinting
Nucleic acid dot/slot blots
Gene probe assays
Colony/plaque lifts
Replica plating
Also suited for Gene probe assays
Dot/slot blots
Replica plating
Advantages High sensitivity
Low background
Highest sensitivity
Positive charge over
wide pH range
Binding Interaction Hydrophobic and
electrostatic
Hydrophobic and
electrostatic
Nucleic Acid Fixation Bake and UV crosslink Bake and UV crosslink
Detection Methods Radiolabeled probes
Enzyme-antibody
conjugates
Chemiluminescent and
chromogenic substrates
Radiolabeled Probes
Enzyme-antibody
conjugates
Chemiluminescent and
chromogenic substrates

 

 

Product Biodyne Plus Membrane Biodyne C Membrane
Description Positively-charged Nylon 6,6 Negatively-charged Nylon 6,6
Recommended for DNA and RNA transfers
Multiple reprobes
Nucleic acid dot/slot blots
Colony/plaque lifts
Replica plating
Reverse dot blots
Also suited for DNA fingerprinting Protein immobilization
Affinity purification
ELISA
Advantages High sensitivity
Low background
Positive charge
Negative charge over
wide pH range
Surface carboxyl groups
can be derivatized
Binding Interaction Hydrophobic and
electrostatic
Hydrophobic and
electrostatic; covalent
after derivatization
Nucleic Acid Fixation Bake and UV crosslink Not needed after
derivatization
Detection Methods Radiolabeled probes
Enzyme-antibody
conjugates
Chemiluminescent and
chromogenic substrates
Enzyme-antibody
conjugates
Chemiluminescent and
chromogenic substrates

 

 

Product BioTrace NT Membrane BioTrace PVDF Membrane
Description Pure Nitrocellulose Polyvinylidene Fluoride
Recommended for Protein transfers Protein transfers
Also suited for Nucleic acid detection
ELISA
Protein dot or slot
blots
Advantages High sensitivity
Low background
Highest sensitivity
Low background
Chemical resistance
High tensile strength
Binding Interaction Hydrophobic and
electrostatic
Hydrophobic
Method of Immobilization Western transfer
Dot or slot blot
Bake and UV crosslink
(for nucleic acids)
Western transfer
Dot or slot blot
Detection Methods Radiolabeled probes
Direct stains
Fluorescence
Enzyme-antibody
conjugates
Chemiluminescence
Chromogenic
Direct stain
Enzyme-antibody
conjugates
Chemiluminescence
Chromogenic

 

 

Product FluoroTrans Membrane FluoroTrans W Membrane
Description Polyvinylidene Fluoride Polyvinylidene Fluoride
Recommended for N terminal protein
sequencing
Protein transfers
Also suited for Protein transfers
with immunodetection
Protein dot or slot
blots
Advantages High binding capacity
and bond strength
Lowest burn through
Chemical resistance
High tensile strength
Highest sensitivity
Low background
Low burn through
High bond strength
Chemical resistance
High tensile strength
Binding Interaction Hydrophobic Hydrophobic
Method of Immobilization Western transfer
Dot or slot blot
Western transfer
Dot or slot blot
Detection Methods Enzyme-antibody
conjugates
Chemiluminescence
Chromogenic
Direct stain
Enzyme-antibody
conjugates
Chemiluminescence
Chromogenic

 

 

Product UltraBind Membrane
Description Modified Polyethersulfone
Recommended  for ELISA
Affinity separations
Also suited for Hybridoma screening
Advantages No preactivation required
High binding capacity
Binding Interaction Covalent
Method of Immobilization Dot or slot blot
Detection Methods Enzyme-antibody conjugates
Chemiluminescence
Chromogenic

 

 

General Suggestions for Membrane Handling 

  • To avoid contamination, always wear gloves and use blunt-ended forceps (Pall Life Sciences Part Number 51147) when handling membranes.
  • If needed, cut membranes to size while it is between the layers of interleaving material.
  • A clean, sharp pair of scissors or a scalpel should be used to cut the membrane.
  • To wet Pall PVDF and NT membranes:BioTrace NT membrane: Starting at one end, slowly lower the membrane at a 45-degree angle into the wetting buffer, allowing it to wet out slowly and completely. Do not rapidly submerge the membrane, as incomplete wetting may result. Agitate gently for 5 to 10 minutes. If the membrane does not wet out completely, add sufficient methanol to the wetting buffer to achieve a final concentration of 20% (v:v) (20 mL of methanol/80 mL wetting buffer).

    PVDF membranes (FluoroTrans, FluoroTrans W and BioTrace PVDF membranes): Slowly lower the membrane into 80 to 100% (v:v) methanol or ethanol and agitate briefly. The membrane will become translucent as it wets. Rinse the membrane with deionized or distilled water and equilibrate in transfer buffer for 5 minutes prior to use. Do not allow the membrane to dry out.

    (Biodyne membranes do not require wetting prior to use.)

 

Solutions for Nucleic Acid Transfers

Solution Description
Depurination Solution 0.25 M HCl (19 mL 13.3 M HCl/L H2O)
High Salt Denaturation Solution 0.5 M NaOH, 1.5 M NaCl (20 g NaOH, 87.7 g NaCl/L H2O)
Alkaline Transfer Buffer 0.4 N NaOH (16 g/L H2O, filter prior to use)
Neutralizing Solution 0.5 M Tris-HCl pH 7.4, 1.5 M NaCl
20X SSC 3 M NaCl, 0.3 M Sodium Citrate, pH 7.0 (175.3 g NaCl, 88.2 g Sodium Citrate. Dissolve in 800 mL H2O and adjust pH to 7.0 with NaOH or HCl. Add H2O to 1 L.)
10X SSC 1.5 M NaCl, 0.15 M Sodium Citrate, pH 7.0 (1:2 dilution of 20X SSC)
20X SSPE 3.6 M NaCl, 0.2 M Sodium Phosphate, pH 7.7, 0.02 M EDTA (Dissolve 174 g NaCl, 27.6 g NaH2PO4. H2O and 7.4 g EDTA in 800 mL H2O. Adjust pH to 7.7 with NaOH. Add H2O to 1 L.)
50% PEG Polyethylene Glycol, mw 8,000, 10 g in 20 mL sterile water
20% SDS 20 g Sodium Dodecyl Sulfate (SDS) dissolved in 100 mL sterile H2O
Digestion Solution 50 mM Tris/HCl, pH 7.6, 0.1% SDS, 50 mM NaCl, 100 µg/mL Proteinase K (50 mL 1M Tris, 10 mL 10% SDS, 2 mL 5 M NaCl/L. Add Proteinase K just prior to use.)

 

Nucleic Acid Transfer Procedures

DNA Dot Blots with Biodyne MembranesWe recommend fixation by baking at 80°C for 30 minutes followed by UV exposure for all Biodyne membranes (see Fixation Procedures for Biodyne Membranes below).

  1. Denature the DNA. Solubilize it in 6X SSC, heat to 95 °C for 10 minutes, and quickly chill on ice. (Alternatively, the DNA can be denatured by solubilizing it in 0.4 N NaOH.)
  2. Apply the DNA (0.1 pg to 1 ng per spot/slot) to the membrane using a slot/dot blot manifold according to the manufacturer’s instructions. If the total volume per spot is <= 2 µL, a micropipettor can be used to apply each spot. (50 to 75 µL per well works well if a manifold is used. There should be at least 1 pg of sample per well to expect a positive response, although many systems will detect as little as 30 fg of sample.)
  3. Fix the DNA to the membrane by baking for 30 minutes at 80 °C followed by exposure to 80 to 150 mJ of UV light. (If the UV light source is not calibrated, perform a pilot experiment to determine optimal exposure.) Overexposure can result in a loss of sensitivity.
  4. Rehydrate the membrane by incubating in 2X SSC (0.5 mL/cm2) for 5 minutes at room temperature.
  5. For Hybridization and Detection Conditions in next section below.

Note: Dot blots can also be performed without a manifold. DNA can be applied to the membrane with a micropipet or transfer pin tool.DNA Transfers (Southern Blot) with Biodyne Membranes

Most detection systems yield the best results with Biodyne membranes using the following procedure (Pall Improved Alkaline Transfer). It differs from standard protocols in that the gel is not neutralized after the denaturation step. Thus, the initial stages of the transfer occur at high pH, promoting attachment of the DNA to the membrane. A neutral transfer buffer is used, which progressively lowers pH during the transfer, stabilizing the immobilized DNA on the membrane. Good results are also achieved using published standard procedures.

Perform electrophoresis and prepare the gel for transfer:

  1. Load the restriction-digested DNA in sample buffer containing bromophenol blue and electrophorese in an agarose gel.
  2. When electrophoresis is complete, depurinate the DNA by incubating the gel briefly (< 20 minutes) in 2 volumes of 0.25 M HCl. Brief incubation times reduce the size of the DNA fragments and facilitate transfer; excessive incubation times may generate DNA fragments that are too small to hybridize with the probe. (This step is not necessary if the DNA being transferred is less than 4 Kb.)
  3. Rinse the gel in distilled water to remove excess HCl.
  4. Incubate the gel in 2 volumes of High Salt Denaturation Solution. The bromophenol blue dye should revert to blue color; if it does not, repeat this step with fresh Denaturation Solution.

 

Pall Improved Alkaline Transfer

  1. Remove the gel from the High Salt Denaturation Solution and place it on a supported wick of paper (see Figure 2). Assure that the ends of the wick are in the transfer buffer
  2. Place a dry piece of Biodyne membrane directly on the gel.
  3. Cover the membrane with three sheets of blotting paper and a stack of blotting pads. Note: Pads ensure more even liquid flow than paper towels across the membrane.
  4. Place a cover (a plastic or glass plate works well) over the pads and put a small (~ 300 g) weight on the cover.
  5. Allow the transfer to proceed for 4 to 18 hours. While transfer of most fragments may occur in less than 4 hours, minimum transfer times need to be determined empirically and can vary based on agarose concentration in the gel and DNA fragment size.

RNA Transfers (Northern Blots) with Biodyne Membranes

The following procedures are suitable for Biodyne A, B, and Plus membranes. Biodyne B membrane is recommended for detection systems using 32P-labeled probes.

Note: When working with RNA, it is important to keep the equipment and reagent solutions free from RNAse contamination. It is recommended that 0.1% SDS is incorporated in all solutions except the 20X SSC transfer buffer. Water used to prepare buffers can also be treated with 0.1% diethylpyrocarbonate to inactivate RNAse.

  1. Separate the formamide denatured RNA in a formaldehyde agarose gel.
  2. Neutralize the gel by soaking it in 2X SSC for 10 minutes.
  3. Assemble the capillary transfer apparatus as shown in Figure 2 above. Use 20X SSC as the transfer buffer, and follow the instructions for the Pall Improved Alkaline Transfer (steps 1-4). Overnight transfer is recommended to ensure efficient transfer of large RNA molecules.
  4. Remove the membrane from the gel surface. (Do not rinse the membrane.)
  5. Fix the RNA to the membrane by baking for 30 minutes at 80 °C followed by exposure to 80 to 150 mJ of UV light. (If the UV light source is not calibrated, perform a pilot experiment to determine optimal exposure.) Overexposure can result in a loss of sensitivity.

Colony/Plaque Lifts and Replica Plating with Biodyne A Membrane

1.2 µm Biodyne A membrane is recommended for colony and plaque lifts while 0.2 µm Biodyne A membrane is recommended for replica plating. To avoid contamination, prepare the membranes as follows:

  1. Boil the membranes in 1 mM EDTA for 5 minutes and thoroughly rinse with distilled water.
  2. Interleaf the membranes with chromatographic filter paper and wrap in aluminum foil; autoclave for 20 minutes and vacuum dry.

Colony and Plaque Lifts

  1. Grow bacterial colonies or phage plaques on agar plates until they reach the appropriate size (usually just visible). It is important not to overgrow the colonies or plaques.
  2. Cool the plates at 4 °C for 30 minutes.
  3. Hold the membrane disc with blunt-ended forceps (Pall Life Sciences PN 51147) and carefully lay it onto the agar surface.
  4. Mark the orientation of the membrane with a pen or needle.
  5. Prepare two trays containing absorbent paper; one saturated with High Salt Denaturation Solution and one saturated with Neutralizing Solution.
  6. Carefully remove the membrane disc in a single movement and place on filter paper saturated with High Salt Denaturation Solution (colony or plaque side up) for 5 minutes.
  7. Remove the membrane disc and place briefly (colony side up) on dry filter paper to remove excess fluid. Neutralize by placing it on filter paper saturated in Neutralizing Solution for 15 minutes.
  8. Fix the DNA on the membrane.
  9. After fixation, remove proteins by incubating the membrane in Digestion Solution for 6 to 12 hours at 37 °C. Yellow-colored colony spots will disappear during digestion.
  10. Rinse the membrane in 2X SSC and proceed with hybridization and detection.

Replica Plating and Amplification with Biodyne A Membrane

Bacterial colonies can be grown directly on Biodyne A membrane that has been pretreated and autoclaved as described above. Membrane replicas can be frozen for long-term storage.

  1. Using aseptic technique, carefully lay the membrane disc on the surface of the agar plate. Take care to ensure that no air bubbles are trapped between the membrane and the agar.
  2. Prepare an appropriate dilution of the bacterial suspension and spread directly on the membrane. (Use 0.5 mL for 82 mm discs and 1.2 mL for 132 mm discs.)
  3. Incubate at 37 °C until small (< 0.5 mm) colonies are visible.
  4. Remove the membrane disc and place it (colony side up) on sterile filter paper on a glass plate.
  5. Position a second disc of membrane exactly over the first, laying it down in a single movement. Do not attempt to realign the membrane or colonies will be blurred.
  6. Cover with a second piece of sterile filter paper and a glass plate. Apply strong hand pressure to the upper glass plate.
  7. Mark the orientation of the membranes using a hot needle, piercing both membranes while they still adhere together.
  8. Carefully separate the membranes and place each (colony side up) on a fresh agar plate.
  9. The original colonies regrow on the first membrane disc; store this plate at 4 °C. Colonies on the new replica may take slightly longer to grow (4 to 6 hours).
  10. Once colonies on the replica reach an appropriate size, process the membrane as for colony and plaque lifts (above).

This technique can also be used to prepare microarrays using a pin spotting tool.

Fixation Procedures for Biodyne Membranes

All Biodyne membranes should be fixed by baking for 30 minutes at 80 °C followed by exposure to 80 to 150 mJ of UV light. (If the UV light source is not calibrated, perform a pilot experiment to determine optimal exposure.) Overexposure can result in loss if sensitivity. After fixation, proceed with hybridization and detection.

 

Hybridization Procedures for Biodyne Membranes

Pall Life Sciences membranes have been used successfully with all commercially-available DNA detection kits. Manufacturer’s recommendations should be followed with each kit. Additional information may be obtained from either the kit manufacturer’s technical support staff or from Pall Life Sciences.Prehybridization

Prehybridization and hybridization procedures will vary with the type and source of probe DNA. Prehybridization (blocking non-specific binding sites on the membrane) is not required with all detection systems. Tests at Pall have shown that the most important components in a prehybridization solution are SDS and casein (Hammersten grade, BDH, Cat # 44020 is recommended). Prehybridization can be performed for 30 minutes to several hours. In general, higher temperatures (up to 68 °C) will result in faster and more efficient prehybridization.

 

Hybridization

As with prehybridization, hybridization will vary according to the nature of probe DNA and the detection system used. Procedures routinely used with 32P- labeled probes are described below. The following solutions and procedures follow recommendations made by the United States Federal Bureau of Investigation (F.B.I.). No prehybridization step is required.

Hybridization Buffer:

20.4 mL sterile water
12.0 mL 50% PEG (mw 8,000)
4.5 mL 20X SSPE
21.0 mL 20% SDS
57.9 mL total

  1. Add denatured probe and bring to a total volume of 60 mL with sterile water. Use a final concentration of 5 X 105 dpm/mL of labeled probe.
  2. Add 0.1 mL/cm2 of hybridization solution to the membranes. Incubate at 65 °C overnight with slow agitation. (If hybridization is being done in bottles, assure that the volume is sufficient to achieve good wetting of the membrane, typically 10 to 15 mL/bottle.)

Alternative procedure for Hybridization

  1. Prehybridize the membrane by sealing it in a plastic bag with hybridization solution (2 to 4 mL/100 cm2 membrane). Incubate for 30 minutes at 65 °C (DNA transfers) or 42 °C (RNA transfers).
  2. Denature the labeled probe by boiling for 5 minutes followed by snap cooling for 1 minute.
  3. Add sufficient denatured labeled probe to yield 2 mL hybridization solution/100 cm2 membrane and a final probe concentration of 1 to 10 pg/mL.
  4. Remove the membrane from the prehybridization bag and place in a fresh bag with the hybridization solution containing the denatured probe. (Hybridization in bottles also works well with Church’s buffer: 7% (v:v) SDS, 250 mM sodium phosphate pH 7.4.)
  5. Incubate at 65 °C (DNA transfers) or 42 °C (RNA transfers) for 4 hours to overnight.

Post-hybridization Washes

Place membranes in fresh bags or containers and wash with 2 mL/cm2 as follows:

  1. 2X SSC, 0.1% SDS, 15 minutes at room temperature.
  2. 2X SSC, 0.1% SDS, 15 minutes at room temperature.
  3. 0.1X SSC, 0.1% SDS, 20 minutes at 65 °C. (Prewarm this wash solution prior to use.)
  4. Remove excess liquid by lightly blotting the membrane on blotting paper. Do not allow the membrane to dry out.

Use these wash recommendations as a starting point. Wash times, number of repititions, and stringency may need to be optimized for individual probes. Note that over-washing can reduce sensitivity.

 

If membranes are to be reprobed, do not allow them to dry out at any stage following hybridization. For short-term storage (< 1 month), membranes can be kept between moist sheets of chromatography paper at 4 °C.

Autoradiography

  1. Wrap the damp membranes in clear plastic.
  2. In a darkroom, assemble an autoradiography cassette with the side of the membrane that faced the gel against X-ray or autoradiography film (such as Kodak* XAR or Amersham Hyperfilm* MP). Incubate film cassette at -80 °C. Typical exposures are from 24 to 48 hours.

Reprobing

Biodyne membranes are easily reprobed. The procedure listed below follows recommendations by the F.B.I. for use with 32P-labeled probes. This method can also be used with non-radioactive techniques. In some systems which include protein in the prehybridization, hybridization, or blocking steps, pretreatment with protease (0.5 mg/mL proteinase K, 0.1% SDS, at 65 °C for 60 minutes) may be required to allow the stripping agents access to the hybridized DNA.

Formamide Stripping (DNA Transfers)

Formamide is a hazardous chemical. Health hazards include irritation on contact with skin, eyes, or mucous membranes. Liver injury may occur. Ingestion leads to headache, dizziness, nausea, and vomiting. Store formamide in a cool and dry area away from sources of fire. Use only inside fume hood, with chemical- resistant gloves and safety pipette devices. Carefully review the supplier’s MSDS before proceeding.

This procedure is recommended by the F.B.I. for use with DNA transfers hybridized with 32P-labeled probes.

  1. Incubate membranes in the following solution (20 mL/cm2 membrane) for 60 minutes at 65 °C:
    110 mL formamide (deionized using 10 grams Amberlite MB-1 resisn [Sigma])
    20 mL 20X SSPE
    10 mL 20% SDS
    60 mL sterile water
  2. Rinse the membrane in 0.1X SSC, 0.1% SDS (20 mL/cm2 membrane) for 1 minute at room temperature.
  3. Remove excess liquid by placing the membrane on filter paper for 30 seconds.
  4. If membranes are to be stored after stripping and rinsing, wrap in plastic wrap and store at -80 °C.
  5. Repeat prehybridization and rehybridization steps with the new probe.

Stripping with Boiling SDS

  1. Pour boiling 0.1% aqueous SDS solution onto the membrane and gently agitate for several minutes. Note: If the membrane is to be stripped and reprobed many times, loss of bound nucleic acid can be minimized by heating the SDS solution to 90 °C rather than boiling.
  2. Discard the solution and immediately add fresh boiling 0.1% SDS solution. Allow to cool to room temperature.

Alkaline Stripping

This technique should not be used for RNA transfers. It is recommended for probes that will not strip under other conditions. There may be some loss of target DNA from the membrane with this method.

  1. Incubate the membrane in 0.4 M NaOH at 45 °C for 30 minutes.
  2. Wash the membrane in 0.1X SSC, 0.1% SDS for 15 minutes.

 Formamide Stripping (RNA Transfers)

See warning regarding the use of formamide.

  1. Incubate the wet membrane in 75% (v:v) deionized formamide in 10 mM sodium phosphate buffer (pH 7.5) at 65 °C for 1 hour.
  2. Wash the membranes with 0.1X SSC, 0.1% SDS for 15 minutes at room temperature.
  3. Check for probe removal and rehybridize as for the original probe.

 

 

Troubleshooting Guide – Nucleic Acids

A membrane test strip containing a positive control will help determine where the problem lies. If a detection kit is being used, refer to the insert for additional troubleshooting suggestions. 

Problem Likely Causes Possible Remedies
Low sensitivity
(signal intensity)
Poor probe labeling efficiency – Repeat with freshly-made probe
– Use high specific activity nucleotides
Probe concentration too low Increase probe concentration in the hybridization solution
Wash conditions too stringent Increase ionic strength of wash buffer or decrease wash temperature
Inefficient DNA transfer Run extra lanes on the gel and verify transfer by staining with Ethidium Bromide
DNA not retained by the membrane Verify that fixation procedures are appropriate (i.e., no overexposure to UV light)
Incomplete probe denaturation Ensure that probe is completely heat denatured and quick chilled
Detection/exposure time too short Increase autoradiography (or color development) time or increase concentration of enzyme conjugate (non-radioactive methods)
Background spots Particulate in buffers Filter reagents prior to use
High background Probe concentration too high Decrease probe concentration
Insufficient blocking/prehybridization Increase concentration of blocking agent (casein and/or SDS) or prehybridization time
Membrane dried out during processing Keep the membrane wet during all processing steps
Probe “dirty” (unincorporated nucleotides present) Purify probe using a Nanosep® device (call Pall Life Sciences technical service for procedure)
Wash stringency too low Increase wash temperature or decrease ionic strength
Particulate present in hybridization buffer – Filter buffers prior to use
– Wear only powder-free gloves
High background after reprobing Membrane dried out Keep the membrane wet during all processing steps
Membrane not sufficiently reblocked/prehybridized Block or prehybridize prior to each hybridization
Probe not stripped from the membrane Membrane dried out – Probe may not be able to be fully stripped if
membrane has dried out
– Keep membrane moist
Stripping procedure not stringent enough Increase stripping temperature or time, or increase concentration of detergent of formamide
Lack of sensitivity on reprobing Insufficient fixation after transfer – Make sure that the proper fixation procedure
for the membrane is used
– Use Biodyne B membrane
Low (second) probe activity Confirm specific activity of all probes prior to use
Areas on membrane
missing signal
Air bubbles between membrane and gel Eliminate bubbles between membrane and gel by gently rolling a pipette over the surface

 

 

Solutions for Protein Transfer/Immobilization

Solution Description
Towbin Transfer Buffer 25 mm Tris-HCl pH 8.3, 192 mM Glycine, 20% (v:v) Methanol
CAPS Transfer Buffer Dissolve 2.2 g CAPS (3-[cyclohexylamino]-1 propane sulfonic acid) in
100 mL of methanol. Add 900 mL of distilled water and mix well. The pH should be 10.5.
TBS 10 mM Tris-HCl pH 8.0, 150 mM NaCl
TBS-T 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.12% Tween* 20
Reaction Buffer I 10 mM Tris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2
BCIP/NBT (Bromochloroiodyl
phosphate/Nitrobule tetrazolium) Substrate
Dissolve 82.5 mg BCIP, 42.5 mg NBT in 1 mL dimethylformamide. Add, while stirring, to 250 mL Reaction Buffer I. Protect from light.
Reaction Buffer II 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20
DAB (3,3′-Diaminobenzidine
HCl) Subtrate
0.05% DAB in Reaction Buffer II made fresh prior to use
30% Hydrogen Peroxide 30% (v:v) H2O2 in water
CN (4-chloro-1-naphthol) Substrate Dissolve 100 mg/mL CN in methanol
Amido Black Stain Add 45 mL Methanol, 2 mL Acetic Acid, 53 mL Distilled water, 0.1 g Amido Black
Destaining Solution Add 180 mL Methanol, 4 mL Acetic Acid, 16 mL Distilled water
PBS-T 10 mM Sodium Phosphate pH 7.4, 120 mM NaCl, 2.7 mM KCl, 0.3% Tween 20
Colloidal Gold Solution Example: Nanogold* labeling reagents available from Nanoprobes, Inc. (Stony Brook, NY)
Ponceau S Solution Dissolve 0.5 g Ponceau S in 1 mL glacial acetic acid. Add water to a final volume of 100 mL.

 

Protein Transfer and Immobilization

 Protein Transfers (Western Blots) with BioTrace NT and PVDF MembranesPall PVDF and nitrocellulose membranes are recommended for protein transfer and detection. The procedures outlined below are intended as general guidelines; the transfer apparatus manufacturer’s instructions should be followed when assembling the transfer tank. These membranes will provide excellent results with commercially available nonradioactive detection kits using the manufacturer’s published procedures.

Transfer Procedure (FluoroTrans, FluoroTrans W, BioTrace PVDF and BioTrace NT membranes)

  1. If the membranes and absorbent pads are not precut to size, cut them to the size of the gel. Always wear gloves, handle the membrane with blunt-ended forceps (Pall Life Sciences PN51147), and cut the membrane while it is between sheets of the interleaving material.
  2. Wet the membrane according to the procedure.
  3. Equilibrate both the gel and membrane in transfer buffer.
  4. Saturate six new absorbent pads (cut to size if needed) in transfer buffer (or use the number of pads recommended by the apparatus manufacturer). Place three pads on the anode (+) plate.
  5. Carefully place the membrane on the saturated pads. Roll a clean glass pipette slowly and gently over the membrane in one direction to eliminate air bubbles that may exist between the pads and the membrane.
  6. Place the gel on top of the membrane, rolling a glass pipette slowly and gently over the gel in one direction to eliminate air bubbles that may exist between the gel and membrane.
  7. Place three absorbent pads on top of the gel, then place the cathode side (-) of the apparatus on top of the stack.
  8. Insert the stack in the tank and add transfer buffer per the manufacturer’s instructions.
  9. Connect the tank to the power supply and start the transfer. Follow the manufacturer’s recommendations for current. Transfers are generally complete in 15 to 90 minutes.
  10. For BioTrace NT membrane: After the transfer is complete, allow the membrane to air dry at room temperature. This helps the proteins to bind more strongly to the membrane, preventing loss during subsequent washes and detection steps.
  11. For PVDF membranes (FluoroTrans, FluoroTrans W, and BioTrace PVDF): Do not allow the membrane to dry out at any point during the detection. (If the membrane becomes partially dry, allow the membrane to dry fully, then re-wet with methanol and exchange to buffer before continuing.)

General Procedure for Immunodetection

  1.  Block non-specific binding by using either a commercial blocking agent or one of the following blocking solutions:

2% nonfat dry milk10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1-5% BSA

10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% casein

10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20 (for BioTrace NT membrane only)

Note: BSA (1 to 5%) may be used as a blocking agent for nitrocellulose membranes, but is not effective when using PVDF membranes. Casein provides superior blocking performance with all membrane types.

  1. Immerse membrane in blocking agent and incubate with gentle agitation for 1 hour.
  2. Dilute the primary antibody in blocking buffer. Incubate the membrane in this solution for 1 hour with gentle agitation.
  3. Remove unbound antibody by washing the membrane with TBS-T, 3 X 5 minutes at room temperature.
  4. Dilute the secondary labeled antibody (e.g., radioactive or enzyme conjugated) in TBS. Incubate the membrane in this solution at room temperature for 45 minutes to 1 hour with gentle agitation.
  5. Remove unbound antibody by washing the membrane with TBS-T, 3 X 5 minutes at room temperature.
  6. Wash the membrane in TBS, 3 X 5 minutes at room temperature.

Detection with Alkaline Phosphatase and BCIP/NBT

  1. Equilibrate the membrane in Reaction Buffer I at room temperature for 5 minutes.
  2. Remove the buffer and add BCIP/NBT Substrate. Observe the reaction as color develops.
  3. When the reaction is complete, rinse the membrane twice with reaction buffer.
  4. Rinse the membrane twice with distilled water.

Detection with Horseradish Peroxidase and DAB

DAB is a carcinogen and should be handled with appropriate safeguards. Carefully review the supplier’s MSDS before proceeding.

  1. Equilibrate the membrane in Reaction Buffer II at room temperature for 5 minutes.
  2. Add 3 µL of 30% H2O2 to 100 mL of DAB Substrate.
  3. Remove the membrane from the reaction buffer and add the above DAB solution.
  4. Gently agitate at room temperature and observe the reaction as color develops.
  5. Rinse the membrane twice with distilled water.

Detection with Horseradish Peroxidase and Chloronaphthol

  1. Equilibrate the membrane in Reaction Buffer II for 5 minutes at room temperature.
  2. Add 50 µL of CN Substrate to 2 mL methanol. Add 8 mL TBS and mix. Add 35 µL of 30% H2O2and mix again.
  3. Remove the membrane from the reaction buffer, blot briefly on blotting paper, and immerse it in the above solution.
  4. Gently agitate at room temperature and observe the reaction as color develops.
  5. When the desired intensity is achieved, stop the reaction by rinsing the membrane several times in distilled water.

Amido Black Staining

  1. When transfer is complete place the membrane in a tray containing 50 mL of stain solution (do not allow the membrane to dry).
  2. Incubate in stain solution with agitation at room temperature for 4 minutes.
  3. Replace stain solution with destain solution. Incubate for 5 minutes with agitation at room temperature.
  4. Rinse membrane in deionized water.
  5. Air dry.

Colloidal Gold Staining

  1. Incubate the membrane in PBS-T with gentle agitation, 2 X 30 minutes at room temperature.
  2. Rinse the membrane briefly with distilled water.
  3. Seal the membrane and approximately 50 mL of Colloidal Gold in a bag.
  4. Incubate with gentle agitation for 1 hour.
  5. Remove the membrane from the bag, rinse briefly with distilled water, and allow to air dry.

Coomassie Blue Staining

FluoroTrans W and BioTrace PVDF membranes:

  1. Rinse membrane in deionized water.
  2. Stain in 0.2% Coomassie Blue (R250), 40% methanol, 10% acetic acid, for 10 minutes.
  3. Destain I: 80% methanol, 10% acetic acid, for 10 minutes.
  4. Destain II: 45% methanol, 10% acetic acid, for 1 hour. Change solution. Incubate overnight.
  5. Air dry.

The procedures above allow for a visual assessment of transfer efficiency by staining the membrane after transfer is complete. Alternatively, proteins may be transferred after staining the gel with coomassie blue or other total protein stain. If proteins are transferred from a stained gel, the membrane should be destained overnight with three changes of methanol prior to proceeding with detection.

Ponceau S Reversible Staining

FluoroTrans W, BioTrace PVDF, and BioTrace NT membranes:

  1. Place membrane in 0.01% Ponceau S, 3% Trichloroacetic acid solution at room temperature for 1 minute (BioTrace NT membrane) or 5 minutes (FluoroTrans W or BioTrace PVDF membrane).
  2. Wash in water for 2 minutes to destain the membrane.
  3. Wash an additional 10 minutes to remove all the stain.

 

Troubleshooting Guide – Protein Transfers

Problem Likely Cause(s) Possible Remedies
Low sensitivity or absent signal Low antibody activity or titer
  • Aliquot and store antibody solutions at -20 °C and avoid multiple freeze/thaw cycles
  • Use a higher concentration of primary antibody
Incomplete protein transfer
(confirm by staining gel after transfer)
  • Increase transfer time
  • Decrease concentration of methanol in transfer buffer (if using PVDF membrane)
  • Addition of 0.1% SDS can aid transfer of large proteins
Protein not binding to membrane
  • Assure that there are no air bubbles between the gel and membrane
  • Use a transfer buffer without SDS
  • If using BioTrace™ NT membrane, add methanol to the transfer buffer
Low conjugate activity
  • Store conjugates at 4 °C
  • Increase conjugate concentration or incubation time
Blocking agent interferes with antibody binding
  • Use a different blocking agent
High background throughout membrane Color development reaction too long
  • Stop reaction immediately when desired intensity is achieved
Poor quality antibody-enzyme conjugate
  • Use affinity-purified secondary antibody
Incomplete blocking
  • Increase incubation time
  • Use a different blocking agent
Phosphatase or peroxidase activity present in blocking agent
  • Use a different or commercial blocking agent
Background spots Particulate present in buffers
  • Filter reagents prior to use
Spurious bands or spots on membrane Cross-reactivity of primary antibody
  • Further purify or preadsorb antibody
  • Use a monoclonal (if available)
  • Decrease primary antibody concentration
Phosphatase or peroxidase activity in sample (confirm by omitting primary and secondary antibodies during detection)
  • Inactivate activity by heating blot at 80 °C for 20 minutes prior to blocking

Activation of Biodyne C Membrane

Activation of Biodyne C membrane utilizes cyanuric chloride, dicyclohexylcarbodiimide, or glutaraldehyde. If methylene chloride is used as a solvent, the procedure must be performed beneath a fume hood. Membranes activated in methylene chloride should be protected against exposure to water or water vapor until use. In addition, glutaraldehyde can be added to the membranes in water or buffer (PBS).

All incubation and wash steps should be performed in glass containers or trays. All steps, including weighing of reagents, should be performed in hood. This protocol is covered by U.S. Patent 4,693,985. The purchase of Pall Life Sciences membranes provides you with a license under these patents.

 

Reagents required:
These reagents can be hazardous and should be used in a fume hood. Review the supplier’s MSDS before proceeding.

  • Cyanuric Chloride (Trichloro s triazine, or TsT)
  • Methylene Chloride (MeCl2)
  • Triethylamine (TEA)
  • Dicyclohexylcabodiimide (DCCD)
  • Phosphate Buffered Saline (PBS)
  • Glutaraldehyde

Cyanuric Chloride Activation

  1. Cut the membrane to the desired size. [Always wear gloves and use blunt-ended forceps to handle the membrane (Pall Life Sciences PN 51147).]
  2. Combine 4.3 mL TEA and 65 mL MeCl2. Soak the membrane in this solution for 2 minutes.
  3. Add 2 g TsT and incubate for 15 minutes at room temperature with gentle agitation.
  4. Wash membrane in MeCl2, 4 X 5 minutes (50 mL/wash).
  5. Air dry the membrane. Store the activated membrane in a vacuum dessicator until ready to use.

DCCD Activation

  1. Cut the membrane to the desired size. [Always wear gloves and use blunt-ended forceps to handle the membrane (Pall Life Sciences PN 51147).]
  2. Prepare a 10% (w:v) solution of DCCD in MeCl2 (5 g DCCD in 50 mL MeCl2). Soak the membrane in this solution for 30 minutes at room temperature.
  3. Wash membrane in MeCl2, 4 X 5 minutes (50 mL/wash).
  4. Air dry the membrane. Store the activated membrane in a vacuum dessicator until ready to use.

Glutaraldehyde Activation

  1. Cut the membrane to the desired size. [Always wear gloves and use blunt-ended forceps to handle the membrane (Pall Life Sciences PN 51147).]
  2. Soak the membrane in 50 mL of 0.5 to 5% (v:v) glutaraldehyde in PBS for 2 hours with gentle agitation.
  3. Wash the membrane in PBS, 4 X 5 minutes (50 mL/wash).
  4. Air dry the membrane. The membrane can be stored in a vacuum dessicator or in PBS until use.

 

 

Immobilization of Proteins to UltraBind Membrane

The active aldehyde groups of UltraBind membrane react with free amino groups allowing covalent immobilization of proteins, peptides, or other amino-containing compounds. Special reaction conditions are not required, but buffers containing primary amines or their salts (e.g., Tris or glycine buffers) should be avoided as they contain functional groups that can react with the aldehyde groups on the membrane, interfering with attachment. Inert fillers that increase the viscosity of the immobilization solution (such as polyethylene glycol) will also adversely affect the immobilization reaction.Immobilization by Spot Wetting

  1. Spot the protein (2 mg/mL or higher concentration) onto the membrane with a micropipettor or ink jet sprayer. The volume per spot should be 1 to 5 µL. [We suggest a phosphate-based buffer such as 0.1 M PBS, pH 7.5. Other buffers (pH 4.5 to 10.0) can also be used.] Note: The buffer must not contain primary amines or their salts.
  2. Allow the membrane to air dry completely at room or slightly elevated temperature. This typically takes 1 to 2 minutes. It is critical that the immobilized protein is completely dried onto the membrane before proceeding further.

Immobilization by Perfusion (Slot Blotting)

  1. Blotting solutions should contain 0.1 to 1 mg/mL protein.
  2. Assemble the membrane into slot blotter according to manufacturer’s recommendations.
  3. The total volume per well should be 0.1 mL or more. Adjust the volume so that the flow-through time is 5 minutes or longer. [We suggest a phosphate-based buffer such as 0.1 M PBS, pH 7.5. Other buffers (pH 4.5 to 10.0) can also be used.] Note: The buffer must not contain primary amines or their salts.
  4. Allow the membrane to air dry completely at room or slightly elevated temperature. This typically takes 1 to 2 minutes. It is critical that the immobilized protein is completely dried onto the membrane before proceeding further.

Recommendations for Blocking and Detection

After immobilization and drying, the active sites on the membrane must be blocked. Suggested blocking solutions (prepared in 10–100 mM PBS, pH ~7.0) include:
0.5% casein
1% nonfat dry milk
2% BSA
1% ovalbumin

  1. Incubate the membranes in the blocking solution for 30 minutes at room temperature. It is not necessary to rinse the membrane after blocking.
  2. Allow the membrane to air dry completely at room or slightly elevated temperature prior to proceeding.
  3. The following precipitating chromogens have been used successfully with horseradish peroxidase conjugates:
    Tetramethyl benzidine (TMB)
    4-Chloro-1-naphthol (CN)
    Diaminobenzidine (DAB)
    Diaminobenzidine with NiCl2
  4. The use of aminoethylcarbazole (AEC) or DAB/CoCl2 substrates for horseradish peroxidase is not recommended as it results in higher background.
  5. The following substrates have been used successfully with alkaline phosphatase conjugates: 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium salt
    BCIP/nitroblue tetrazolium (NBT)

 

Troubleshooting Guide – UltraBind Membrane

Problem Likely Cause(s) Possible Remedies
High background Too much conjugate present Decrease conjugate concentration
Membrane insufficiently blocked Change blocking agent
Increase incubation time or concentration of blocking agent
Wash membrane 0.1% Tween 20 in PBS (pH 7.5) after blocking
Background spots Particulate present in buffers Filter reagents prior to use
Weak color reaction  Conjugate concentration too low Do not prewet membrane
Use recommended immobilization buffer
Residual detergent on membrane Wash membrane prior to addition of substrate
Choice of substrate Use a more sensitive substrate (i.e., DAB instead of CN)


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