Introduction

For amino acid analysis and protein sequencing of small amounts of proteins (as little as 10 pmoles), electroblotting onto polyvinylidene difluoride (PVDF) membranes is ideal. In addition, PVDF membranes can be used for immunoblotting (western blot analysis). While our 0.2 µm PVDF membrane performs well for all of these applications, Invitrolon™ PVDF is a high quality, 0.45 µm PVDF membrane particularly suitable for high sensitivity and low background immunoblotting.

Ordering Information

Sku Name Size Price Qty
LC2005 Invitrolon™ PVDF/Filter Paper Sandwich, 0.45 µm pore size, 8.3 cm x 7.3 cm (for mini gels) 20⁄pack USD 193.00
LC2002 PVDF Pre-cut Blotting Membranes, 0.2 µm pore size 20⁄pack USD 186.00
LC2007 Invitrolon™ PVDF/Filter Paper Sandwich, 0.45 µm pore size, 8.5 cm x 13.5 cm 16⁄pack USD 268.00
WB7103 WesternBreeze® Chromogenic Kit, anti-mouse 1 kit USD 314.00
WB7104 WesternBreeze® Chemiluminescent Kit, anti-mouse 1 kit USD 385.00

Using PVDF Membranes

Introduction         
 
Use the procedure below to prepare PVDF membranes for protein transfer (electroblotting).
 
Basic Procedure 
  

Note that the PVDF membrane is supplied between two pieces of pre-cut 3 MM filter paper. The filter paper may be used as part of the blot ‘sandwich’.
All washes are performed in a shallow dish with constant shaking.

   1. Wet the PVDF membrane in 100% alcohol (methanol, ethanol, or isopropanol).
   2. Drain and equilibrate the membrane and filter paper for 5 minutes in the desired transfer buffer.
   3. Assemble the blot ‘sandwich’ according to the instructions provided by the manufacturer of your blot apparatus and transfer.
   4. After transfer, rinse the PVDF membrane with water and proceed to immunoblotting (see below), staining (see below), or drying.

Immunoblotting
     
If you are using the WesternBreeze® Immunodetection Kit from Invitrogen for immunoblotting, refer to the appropriate WesternBreeze® manual for details on blocking and detection of the protein. For more information on the WesternBreeze® Immunodetection Kit, visit our web site at www.invitrogen.com or contact Technical Service.
If you are using any other immunodetection kit, follow the manufacturers recommendations.
 
Staining 

Stain PVDF membranes using SimplyBlue™ SafeStain, Coomassie® Blue R, Amido Black, or Ponceau S. Refer to the table below for instructions. Use 10-20 ml of each stain.

Stain
Concentration
Staining
Destaining I
Destaining II (Optional)
SimplyBlue
Use as supplied
1-2 minutes*
3 x 1 minute in deionized water
--
Coomassie® Blue R
0.1% in 40% ethanol, 10% acetic acid
10 minutes
10 minutes in 40% ethanol, 10% acetic acid.
10 minutes in 90% ethanol, 5% acetic acid
Amido Black
0.1% in 10% ethanol, 2% acetic acid
10 minutes
10 minutes in 10% ethanol, 2% acetic acid
10 minutes in 90% ethanol, 2% acetic acid
Ponceau S
0.5% in 1% acetic acid
10 minutes
10 minutes in 1% acetic acid
--

*Incubation of dry PVDF membranes in SimplyBlue™ for longer than 2 minutes results in high background.

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Blotting Protocols for Novex® Pre-Cast Gels

Western Blotting
 
Introduction

Instructions are provided below for blotting Novex® Gels using the XCell II™ Blot Module. For more information on the XCell II™ Blot Module, refer to the manual (IM-9051). This manual is available on our Web site at www.lifetech.com or contact Technical Service.
 
Materials Supplied by the User

  • Blotting membranes
  • Filter paper
  • Methanol (if using PVDF membranes)
  • XCell II™ Blot module
  • Appropriate Transfer Buffer
  • NuPAGE® Antioxidant for reduced samples for NuPAGE® gels
  • MagicMark™ Western Protein Standard
  • Deionized water


NuPAGE® Antioxidant

The NuPAGE® Antioxidant is used in the transfer buffer for blotting reduced proteins and prevents the proteins from reoxidizing and maintains the proteins in a reduced state. Proteins are oxidized during blotting at a slower rate in the neutral pH environment of NuPAGE® blotting than in a higher pH blotting system. The major cause of reoxidation during blotting is the oxidizing effect of the anode electrochemistry.
 
NuPAGE® Transfer Buffer

We recommend using the NuPAGE® Transfer Buffer for western transfer of NuPAGE® Gels as the transfer buffer maintains the neutral pH environment established during NuPAGE® electrophoresis.
 
The NuPAGE® Transfer Buffer protects against modification of the amino acid side chains and is compatible with N-terminal protein sequencing using Edman degradation.
 
Preparing NuPAGE® Transfer Buffer
Prepare 1000 ml of 1X NuPAGE® Transfer Buffer using the NuPAGE® Transfer Buffer (20X) as follows:

  Reduced Samples Non-Reduced Samples
NuPAGE® Transfer Buffer (20X)50 ml50 ml
NuPAGE® Antioxidant1 ml--
Methanol100 ml*100 ml*
Deionized Water849 ml850 ml
Total Volume1000 ml1000 ml


*NuPAGE® Transfer Buffer with 10% methanol provides optimal transfer of a single gel in the blot module. If you are transferring two gels in the blot module, increase the methanol content to 20% to ensure efficient transfer of both gels.
 
See recipe of the NuPAGE® Transfer Buffer, if you are preparing your own transfer buffer.
 
Preparing Transfer Buffer For blotting Tris-Glycine, Tricine, and IEF Gels
We recommend using the Tris-Glycine Transfer Buffer. An alternate transfer protocol for IEF gels is provided.
 
If you are performing protein sequencing, alternate transfer buffers are listed.
Prepare 1000 ml of 1X Tris-Glycine Transfer Buffer using the Tris-Glycine Transfer Buffer (25X) as follows:

Tris-Glycine Transfer Buffer (25X)40 ml
Methanol200 ml
Deionized Water760 ml
Total Volume1000 ml


See Recipe for a recipe of the Tris-Glycine Transfer Buffer, if you are preparing your own transfer buffer.
 
For blotting TBE and TBE-Urea Gels

Dilute the 5X TBE Running Buffer to 0.5X with deionized water. See Recipe for a recipe of the TBE Running Buffer, if you are preparing your own transfer buffer.
 
Preparing Blotting Pads

Use about 700 ml of 1X Transfer Buffer to soak the pads until saturated. Remove the air bubbles by squeezing the pads while they are submerged in buffer. Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed.
 
Preparing Transfer Membrane and Filter

Cut selected transfer membrane and filter paper to the dimensions of the gel or use Novex® pre-cut membrane/filter paper sandwiches.

  • PVDF membrane—Pre-wet PVDF membrane for 30 seconds in methanol, ethanol, or isopropanol. Briefly rinse in deionized water, then place in a shallow dish with 50 ml of 1X Transfer Buffer for several minutes.
  • Filter paper—Soak the filter paper briefly in 1X Transfer Buffer immediately prior to use.
  • Gel—Use the gel immediately following the run. Do not soak the gel in transfer buffer.


                                                                                                                                                                                                
Western Transfer Using the XCell II™ Blot Module

Wear gloves while performing the blotting procedure to prevent contamination of gels and membranes, and exposure to irritants commonly used in electrotransfer.

Transferring One Gel

  1. After opening the gel cassette as described, remove wells with the Gel Knife.
  2. Place a piece of pre-soaked filter paper on top of the gel, and lay just above the slot in the bottom of the cassette, leaving the “foot” of the gel uncovered. Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller.
  3. Turn the plate over so the gel and filter paper are facing downwards over a gloved hand or clean flat surface.
  4. Use the Gel Knife to push the foot out of the slot in the plate and the gel will fall off.
  5. When the gel is on a flat surface, cut the “foot” off the gel with the gel knife.
  6. Wet the surface of the gel with transfer buffer and position the pre-soaked transfer membrane on the gel, ensuring all air bubbles have been removed.
  7. Place another pre-soaked anode filter paper on top of the membrane. Remove any trapped air bubbles.
  8. Place two soaked blotting pads into the cathode (-) core of the blot module. The cathode core is the deeper of the two cores and the corresponding electrode plate is a darker shade of gray. Carefully pick up the gel membrane assembly and place on blotting pad in the same sequence, such that the gel is closest to the cathode core (see Figure 1).
  9. Add enough pre-soaked blotting pads to rise to 0.5 cm over rim of cathode core. Place the anode (+) core on top of the pads. The gel/membrane assembly should be held securely between the two halves of the blot module ensuring complete contact of all components.
  10. Position the gel/membrane assembly and blotting pads in the cathode core of the XCell II™ Blot Module to fit horizontally across the bottom of the unit. There should be a gap of approximately 1 cm at the top of the electrodes when the pads and assembly are in place.
  11. Hold the blot module together firmly and slide it into the guide rails on the lower buffer chamber. The blot module will only fit into the unit one way, so the (+) sign can be seen in the upper left hand corner of the blot module. Properly placed, the inverted gold post on the right hand side of the blot module will fit into the hole next to the upright gold post on the right side of the lower buffer chamber.
  12. Place the Gel Tension Wedge so that its vertical face is against the blot module. Lock the Gel Tension Wedge by pulling the lever forward.
  13. Fill the blot module with 1X Transfer Buffer until the gel/membrane assembly is covered in this buffer. Do not fill all the way to the top as this will only generate extra conductivity and heat.
  14. Fill the Outer Buffer Chamber with deionized water by pouring approximately 650 ml in the gap between the front of the blot module and the front of the lower buffer chamber. The water level should reach approximately 2 cm from the top of the lower buffer chamber. This serves to dissipate heat produced during the run.
  15. Place the lid on top of the unit.
  16. With the power turned off, plug the red and black leads into the power supply. Refer to Recommended Transfer Conditions.


Transferring Two Gels in One Blot Module

  1. Prepare 1X Transfer Buffer containing 20% methanol as described
  2. Repeat Steps 1–7 above twice to make two gel/membrane sandwiches.
  3. Place two pre-soaked pads on cathode shell of blot module. Place first gel/membrane assembly on pads in correct orientation, so gel is closest to the cathode core. (See Figure 2).
  4.  Add another pre-soaked blotting pad on top of first membrane assembly.
  5. Position second gel/membrane assembly on top of blotting pad in the correct orientation so the gel is closest to the cathode side.
  6. Proceed with steps 8–13 from Transferring One Gel.
  7. Refer to Recommended Transfer Conditions.


                                                                                                                                                                                                    

Figure 1




Figure 2




Recommended Transfer Conditions


The transfer conditions for Novex® Pre-Cast Gels using the XCell II Blot Module are listed in the table below.
Note: The expected current listed in the table is for transferring one gel. If you are transferring two gels in the blot module, the expected current will double.

 

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Gel
Transfer Buffer
Membrane
Power Conditions
NuPAGE® Novex Bis-Tris Gel
1X NuPAGE® Transfer Buffer with 10% methanol*
0.1% NuPAGE® Antioxidant for reduced samples
Nitrocellulose or PVDF
30 Volts constant for 1 hour
Expected Current
Start: 170 mA
End: 110 mA
NuPAGE® Novex Tris-Acetate Gel
1X NuPAGE® Transfer Buffer with 10% methanol*
0.1% NuPAGE® Antioxidant for reduced samples
Nitrocellulose or PVDF
30 Volts constant for 1 hour
Expected Current
Start: 220 mA
End: 180 mA
Tris-Glycine Gel
Tricine Gel
1X Tris-Glycine Transfer Buffer with 20% methanol
Nitrocellulose or PVDF
25 V constant for 1-2 hours
Expected Current
Start: 100 mA
IEF Gel
1X Tris-Glycine Transfer Buffer with 20% methanol
Nitrocellulose or PVDF
25 V constant for 1 hour
Expected Current
Start: 65-85 mA
 
0.7% Acetic acid pH 3.0
See for details on this alternate transfer protocol
Nitrocellulose or PVDF
10 V constant for 1 hour
Expected Current
Start: 65-85mA
TBE Gel
0.5X TBE Running Buffer
Nylon
30 V constant for 1 hour
Expected Current
Start: 39 mA
End: 35 mA
TBE-Urea Gel
0.5X TBE Running Buffer
Nylon
30 V constant for 1 hour
Expected Current
Start: 39 mA
End: 35 mA
DNA Retardation Gel
0.5X TBE Running Buffer
Nylon
30 V constant for 1 hour
Expected Current
Start: 39 mA
End: 35 mA


*NuPAGE® Transfer Buffer with 10% methanol provides optimal transfer conditions when blotting a single gel in a blot unit. If transferring two gels within a blot unit, increase the methanol content to 20% to ensure even and efficient transfer of both gels.
                                                                                                                                                                                                     
Blotting IEF Gels

Novex® IEF Gels are composed of 5% polyacrylamide and are more susceptible to hydrolysis due to the heat generated with the recommended blotting protocol. The following protocol has been optimized to prevent hydrolysis and effective transfer of basic proteins due to the low pH of the transfer buffer.

  1. Chill 0.7% acetic acid which will be used later for transfer.
  2. After electrophoresis of the gel, equilibrate the gel in 0.7% acetic acid for 10 minutes. Tip:The 5% polyacrylamide gels are more sticky and difficult to handle than higher percentage polyacrylamide gels. To lift the gel from the equilibration solution, submerge the filter paper under the gel while the gel is floating in the equilibration solution. When the gel is in the correct position, lift up the filter paper to attach the gel to the filter paper. This prevents the gel from sticking to the filter paper before it is in the proper position and avoids handling of the gel.
  3. Assemble the gel/membrane sandwich as described previously, except in a reverse order so that the membrane is on the cathode (-) side of the gel. This is the opposite of a typical western blotting protocol, where the negatively charged protein will migrate toward the anode (+) during the transfer.
  4. Transfer for 1 hour at 10 V constant.



Blotting Native Gels

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During SDS-PAGE all proteins have a net negative charge due to the SDS in the sample buffer and the running buffer. Proteins separated during native gel electrophoresis do not have a net charge which may cause problems during the transfer. Some native proteins may have a higher pI than the pH of the Tris-Glycine Transfer Buffer used in standard transfer protocols. Guidelines are provided below to increase the transfer efficiency of native proteins.

 

  • Increasing the pH of the transfer buffer to 9.2 (25 mM Tris Base, 25 mM glycine, pH 9.2), allows proteins with pI below 9.2 to transfer towards the anode electrode
  • Place a membrane on both sides of the gel if you are using the regular Tris-Glycine Transfer Buffer, pH 8.3. If there are any proteins that are more basic than the pH of the transfer buffer, they will be captured on the membrane placed on the cathode side of the gel
  • Incubate the gel in 0.1% SDS for 15 minutes before blotting with Tris-Glycine Transfer Buffer. The small amount of SDS will render enough charge to the proteins so they can move unidirectionally towards the anode and in most cases will not denature the protein


Native proteins may diffuse out of the membrane into the solution during the blocking or antibody incubation steps, as the native proteins tend to be more soluble. To prevent diffusion of the proteins out of the membrane, we recommend fixing the proteins to the membrane by air drying the membrane or incubating the membrane in 5-10% acetic acid for 15 minutes followed by rinsing the membrane with deionized water and then air drying. By performing any of these two fixing methods the proteins will be sufficiently unfolded to expose hydrophobic sites and bind more efficiently to the membrane
 
Alternate Transfer Buffers

The NuPAGE® Transfer buffer (with NuPAGE Antioxidant, if samples are reduced) is the optimal buffer for western transfer of NuPAGE® gels. However, you can use the Tris-Glycine Transfer Buffer (1X) or TBE Transfer Buffer (1/2X) for blotting NuPAGE® gels. The NuPAGE® Antioxidant is less functional if added to the Tris-Glycine and TBE buffers. Carbonate and CAPS transfer buffers are not recommended for blotting of NuPAGE® Novex Pre-Cast Gels. The NuPAGE® Antioxidant is ineffective at pH> 9 and will not work when used with the Carbonate or CAPS transfer buffers.

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Specifications

Refer to the table below for the specifications of each type of PVDF membrane.

>Specifications
>Invitrolon PVDF
>0.2 µm PVDF
>Catalog no.
>LC2005 (20 transfers)
>LC2002 (20 transfers)
>Quantity
>20 membrane/filter paper sandwiches
>20 membrane/filter paper sandwiches
>Pore Size
>0.45 µm
>0.2 µm
>Dimensions
>8.3 cm x 7.3 cm
>8.3 cm x 7.3 cm
>Binding Capacity
>Goat IgG: 294 µg/cm2
>BSA: 131 µg/cm2
>Insulin: 85 µg/cm2
>50-150 µg/cm2 for large, globular proteins
>>150 µg/cm2 for smaller peptides
>Application
>Optimal Western Transfers for Proteins > 10 kDa
>Protein Sequencing
>Amino Acid Analysis
>Other Western Transfers
>Protein Sequencing
>Amino Acid Analysis
>Solid Phase Assay Systems
>Re-probe Characteristics
>Yes
>Yes

 

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