Nitrocellulose Western Transfer Membrane

Product	BioTrace NT Membrane	BioTrace PVDF Membrane	FluoroTrans® PVDF Membrane	FluoroTrans W PVDF Membrane
Description	Pure  Nitrocellulose	Polyvinylidene  Fluoride	Polyvinylidene  Fluoride	Polyvinylidene  Fluoride
Application	Protein transfers Nucleic acid    detection  ELISA	Protein transers Protein dot or    slot blots	N terminal protein    sequencing Protein dot or    slot blots	Protein transfers Protein dot or    slot blots Detection with    immunostaining
Advantages	High sensitivity Low background	Highest sensitivity Low background Chemical    resistance High tensile     strength	High binding    capacity Lowest burn    through Chemical     resistance High tensile     strength Low fluorescence    background	Highest sensitivity Low background Low burn     through High binding     capacity Chemical    resistance High tensile     strenghth
Binding Interaction	Hydrophobic    and electrostatic	Hydrophobic	Hydrophobic	Hydrophobic
Method of Immobilization	Western transfer Dot or slot blot Bake and UV    crosslink (for    nucleic acids)	Western transfer Dot or slot blot	Western transfer  Dot or slot blot	Western transfer  Dot or slot blot
Detection Methods	Radiolabeled   probes Direct stains Fluorescence Enzyme-antibody   conjugates	Direct stains Enzyme-antibody    conjugates Cemiluminescence Chromogenic	Direct stains Enzyme-antibody    conjugates Cemiluminescence Chromogenic Fluorescence	Direct stains Enzyme-antibody    conjugates Cemiluminescence Chromogenic

Protocol for BioTrace NT Nitrocellulose Transfer Membrane

Pall 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, non-radioactive detection kits using the manufacturer’s published procedures.

A. Materials Required

1. BioTrace NT membrane 
2. Phosphate buffered saline (PBS)
3. Non-amine containing buffers
4. Protein blocking buffers
   1. 0.5% Casein in PBS
   2. 1% non-fat, dried milk in PBS
      Tip: The best method for blocking membranes is 0.5% Hammersten grade casein (BDH 44020 or equivalent) diluted in buffer. Add casein to the buffer and heat on a stir plate to 80 °C or until dissolved; do not boil the solution. Cool to 25-40 °C before use.
      
5. Blocking solutions may also contain 0.05% Tween 20 or other non-ionic surfactant which may enhance blocking and will also aid in re-hydrating the membrane if the spotted membrane is stored in a dry state.

B. Handling of the Membrane

1. BioTrace NT membrane should be handled carefully and with gloves. Nitrocellulose membranes can be brittle and may crack or tear, especially when dry.
2. Remove the sheet or roll of membrane from package. Cut to desired dimensions of the SDS-PAGE gel.
   Tip: It is important to number the membranes with a pencil to ensure they can be identified during testing. Ink will run in the Western blot transfer procedure.
3. Slowly lower the membrane into 20% (v/v) methanol or ethanol and agitate briefly. The membrane will become translucent as it wets.
4. Rinse the membrane with high purity water and equilibrate in transfer buffer for 5 minutes prior to use. Do not allow the membrane to dry out during processing. (If membrane does become partially dry, allow to dry fully, re-wet with 20% alcohol, exchange to buffer, and proceed. This extra alcohol step does not usually interfere with detection.)

C. Western Transfer

Proteins should be immobilized on the membrane via electro-transfer or dot/slot blotting with a suitable manifold. The optimum amount of protein for detection usually varies between 1 and 10 μg per band. After transfer, membranes can be rinsed in transfer buffer to remove excess gel fragments.

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 (PN 51147), and cut the membrane while it is between sheets of the interleafing 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 15-90 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.

D. Ponceau S Staining Protocol for Reversible Total Protein Detection

1. Soak membranes in 0.01% (w/v) Ponceau S, 3% (w/v) Trichloroacetic acid for 1 minute.
2. Rinse several times in distilled water.
3. Photograph membrane and remove remaining stain with three changes of Tris buffered saline (20mM Tris, 150mM NaCl, pH7).
4. Proceed to blocking step for immunodetection of specific bands.

E. Immunodetection

1. Block non-specific binding by using either a commercial blocking agent or one of the following blocking solutions.
   1. 2% non-fat, dried milk 10 mM Tris-HCl pH 7.5, 150 mM NaCl
   2. 1-5% BSA, 10 mM Tris-HCl pH 7.5, 150 mM NaCl
   3. 0.5% casein 10 mM Tris-HCl pH 7.5, 150 mM NaCl
   4. 0.05% Tween 20 (for BioTrace™ NT membrane only)
      Tip: BSA (1-5%) 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.
2. Each of the following steps is performed in a suitably-sized container to allow 10 mL of solution to ensure adequate coverage of a 10 x 10 cm membrane area. Plastic bags, boxes, or tubes on a roller mixer may also be used to perform incubation steps. Make sure all NT blots are completely immersed beneath the surface of the liquid. Gently agitate samples during all incubation steps on an orbital shaker.
3. Place the membrane blots in a suitably sized container with suitable blocking solution.
4. Place the container on an orbital shaker and gently agitate for 30 minutes.
   Tip: At this point, the membrane blots may be dried at 37 °C for 10 minutes or air-dried at 20-30 °C for > 30 minutes and stored.
5. Place the membrane blots into a clean, suitable container with primary detection antibody diluted in PBS to 1 μg/mL. Use 2 mL per 10 x 10 cm membrane area. Gently agitate the membrane blots on an orbital shaker for 30 minutes.
6. Replace primary antibody solution in the container with 10 mL wash solution. Wash membrane blots in 2x changes of wash solution, 5 minutes per wash. Gently agitate membrane blots on orbital shaker during wash steps.
7. Replace wash solution with 10 mL of PBS. Gently agitate on orbital shaker for 1 minute.
8. Replace the PBS solution with 2 mL (per 10 x 10 cm area) of the secondary detection conjugate diluted 1/1000 in Blocking Solution.
9. Repeat wash procedure listed above.
10. Replace wash solution with two changes of high purity water. Gently agitate on orbital shaker, 1 minute per change of rinse solution. (Substrate buffer may also be used for this rinse step.)

F. Detection Methods

1. Detection with alkaline phosphatase conjugate and BCIP/NBT substrate.
   1. Equilibrate the membrane in Reaction Buffer I (10 mM Tris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2) at room temperature for 5 minutes.
   2. Remove the buffer and add BCIP/NBT* substrate. (Dissolve 82.5 mg BCIP, 42.5 mg NBT in 1 mL dimethylformamide. Add, while stirring, to 250 mL reaction buffer. Protect from light). Observe the reaction as color develops.
   3. When the reaction is complete, rinse the membrane twice with reaction buffer. Rinse the membrane twice with distilled water. (*Bromochloroiodyl phosphate/Nitrobule tetrazolium)
2. Detection with Horseradish Peroxidase and 3,3-Diaminobenzidine HCl) substrate (DAB).
   CAUTION: 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 (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20) at room temperature for 5 minutes.
   2. Add 0.003 mL of 30% H2O2 to 100 mL of DAB substrate (0.05% DAB in reaction buffer made fresh prior to use).
   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.
3. Detection with Horseradish Peroxidase and Chloronaphthol (CN)
   1. Equilibrate the membrane in reaction buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 5 minutes at room temperature.
   2. Add 0.05 mL of CN substrate to 2 mL methanol. Add 8 mL TBS and mix. Add 0.035 mL of 30% H2O2 and 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.

Application Data for BioTrace™ NT Nitrocellulose Transfer Membrane

An example of a Western blot electro-transfer experiment is shown in Figure 3.2. The resulting pre-stained proteins visible on the back up second membrane layer clearly showed reduced “burn-through” with the BioTrace NT membrane.

Figure 3.2

Less “Burn-Through” with BioTrace NT Nitrocellulose Membranes in Western Blotting Electro-Transfer

Pre-stained proteins were separated on an SDS-PAGE electrophoresis gel and electro-transferred to the indicated nitrocellulose membranes. A double layer of membrane was used, one directly against the gel, and followed by the second layer. Signal intensity on the second layer is indicative of “burn through,” which can lead to loss of signal.

Troubleshooting for BioTrace™ NT Nitrocellulose Transfer Membrane

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 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 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 commerical     blocking agent
Background spots	Particulate present in buffers	Filter reagents prior to use
Spurious bands or spots  membrane antibody	Cross-reactivity of primary    antibody    ___________________________	Further purify on or pre-adsorb 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

Ordering Information for BioTrace™ Nitrocellulose Transfer Membrane

Part Number	Description	Pkg
66593	7 x 8.5 cm sheets	10/pkg
66489	20 x 20 cm sheets	10/pkg
66485	30 cm x 3 m roll	1/pkg

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