Introduction

Western electroblotting is the electrophoretic transfer of separated proteins in a gel to the surface of a thin support membrane matrix, where they are bound and immobilized. Semi-dry blotting is performed with plate electrodes in a horizontal configuration, sandwiching a gel and membrane between sheets of buffer-soaked filter paper that function as the ion reservoir. During electrophoretic transfer, negatively charged molecules migrate out of the gel and move towards the positive electrode, where they are deposited on the membrane. The plate electrodes, separated only by the gel and filter paper stack, provide high field strength (V/cm) across the gel, allowing for very efficient, rapid transfers.

 

The Novex® Semi-Dry Blotter is a semi-dry blotting system that allows you to quickly and easily perform simultaneous western blotting of up to 2 Midi Gels, 4 Mini Gels, or 2 E-PAGE™ gels. It can also be used to perform northern (RNA) and Southern (DNA) blotting. The Novex® Semi-Dry Blotter is designed for fast set-up and ease of use. The platinum-coated titanium anode plate and stainless steel cathode plate provide fast and efficient electroblotting using less transfer buffer than wet (tank) blotting methods.

 

Features


The Novex® Semi-Dry Blotter has the following features:

  • Simple, easy-to-use design
  • Allows simultaneous transfer of 1–2 Midi Gels, 1–4 Mini Gels, or 1–2 E-PAGE™ gels
  • Requires less transfer buffer than wet blotting methods
  • Connector design prevents incorrect cable hookups 


Novex® Semi-Dry Blotter Cables


The Novex® Semi-Dry Blotter cables will fit onto the blotter in only one orientation, to prevent incorrect connection to the electrodes. The black (cathode) cable has a white acrylic safety ring attached to the end to prevent connection to the recessed red (anode) connector.
 
Power Supply Requirements


The Novex® Semi-Dry Blotter is used with an external DC power supply that must:

  • Be isolated from the ground so that the DC output is floating
  • Be equipped with No Load or Open Circuit protection
  • Be able to operate at 20 V


The ZOOM® Dual Power Supply and PowerEase® 500 Power Supply are compatible for use with this blotter. See page vii for ordering information.
 
Blotting Membranes
Invitrogen offers three types of blotting membrane and filter paper sandwiches. Refer to page vii for ordering information.

  • Nitrocellulose for western or Southern blotting
  • PVDF (polyvinylidene difluoride) for western blotting
  • Nylon for Southern or northern blotting


See the following table for more details on each membrane.

 

Membrane
Properties
Applications
Pore size
Reprobing
Nitrocellulose
Most widely used membrane for western blotting
Good binding capacity
Proteins bind to the membrane due to hydrophobic interactions
Protein binding capacity:
80 µg/cm2
Western transfer
Amino acid analysis
Solid phase assay systems
0.2 µm
0.45 µm
No
PVDF
Higher binding capacity than nitrocellulose
Strong hydrophobic character and solvent resistant
Physically stronger than nitrocellulose
Compatible with commonly used protein stains and immunodetection methods
Protein binding capacity: 50-150 µg/cm2
Protein sequencing
Western transfer
Amino acid analysis
Solid phase assay systems
0.2 µm
0.45 µm
Yes
Nylon
Microporous membrane modified with strongly basic charged groups
Ideal for binding negatively charged biomolecules such as DNA and RNA
Low background for enhanced resolution
Membrane is formed around a non-woven polyester fiber matrix which confers high tensile strength, toughness, and flexibility
Southern and northern transfers
Solid phase immobilization
Dry chemistry test strips
Enzyme immobilization
Gene probe assays
0.45 µm
Yes

TOP

Materials Needed

  • Methanol
  • Transfer Buffer (see buffer recipes for details)
  • NuPAGE® Antioxidant (for reduced samples)
  • Blotting membranes:
  • Nitrocellulose or PVDF membrane for western transfers (Nitrocellulose/Filter Paper Sandwiches and Invitrolon™/Filter Paper Sandwiches are available separately from Invitrogen)
  • 4 pieces of 2.5-mm thick Blotting Filter Paper per gel (available separately from Invitrogen), or equivalent pieces of thinner blotting filter paper
  • E-PAGE™ Gels only: E-PAGE™ Blotting Pads (included with gels)
  • Blotting roller (available separately from Invitrogen)
  • Deionized water
  • Shallow trays for equilibrating membranes, gels, filter paper, and blotting pads (Incubation Trays are available separately from Invitrogen)
  • Gel
  • Gel knife

Wear gloves at all times during the entire blotting procedure to prevent contamination of gels and membranes, to avoid exposure to irritants commonly used in electrophoresis and blotting procedures, and to avoid staining the anode plate with oils in your skin.
The platinum-plated titanium anode plate is easily scratched and should be handled with care.
Do not adjust the pH of the Transfer Buffer, as this may alter the conductivity of the solution.
TOP

Ordering Information

Catalog # Name Size List Price (USD) Qty
SD1000 Novex® Semi-Dry Blotter 1 each 1,200.00
NP0006 NuPAGE® Transfer Buffer (20X) 125 mL 22.47
NP0005 NuPAGE® Antioxidant 15 mL 25.40
LC3675 Novex® Tris-Glycine Transfer Buffer (25X) 500 mL 37.80
LC2100 Blotting Roller 1 unit 30.60
LC2102 Incubation Tray, 10 x 14 cm 8 each 121.00

Transfer Buffer for Western Blots

Prepare the appropriate transfer buffer for your gel type from the recipes below. For semi-dry western blotting, the transfer buffer must be at twice the concentration used in wet blotting (i.e., 2X) to ensure that there are enough buffering ions present in the smaller volume of liquid.
 
NuPAGE® Novex® Bis-Tris or Tris Acetate Gels, and E-PAGE™ 48 Gels

Prepare the following 2X NuPAGE® Transfer Buffer. This transfer buffer maintains the neutral pH environment established during gel electrophoresis, protects against modification of the amino acid side chains, and is compatible with N-terminal protein sequencing using Edman degradation.
A volume of 500 ml is sufficient for blotting 1 Midi Gel or 2 Mini Gels.

  Reduced Non-reduced
NuPAGE® Transfer Buffer (20X) 50 ml 50 ml
Methanol 50 ml 50 ml
NuPAGE® Antioxidant                        
0.5 ml
 —
Deionized Water to 500 ml to 500 ml


E-PAGE™ 96 Gels

Prepare the following 2X NuPAGE® Transfer Buffer without methanol. A volume of 500 ml is sufficient for blotting 1 E-PAGE™ 96 gel.


  Reduced Non-reduced
NuPAGE® Transfer Buffer (20X) 50 ml 50 ml
NuPAGE® Antioxidant  0.5 ml
Deionized Water to 500 ml to 500 ml

Novex® Tris Glycine or Tricine Gels

Prepare the following 2X Tris-Glycine Transfer Buffer. A volume of 500 ml is sufficient for blotting 1 Midi Gel or 2 Mini Gels.

Tris-Glycine Transfer Buffer (25X)                  40 ml
Methanol                                                            50 ml
Deionized Water                                             to 500 ml
TOP

Semi-Dry Blotting Procedure

Equilibrating the Gel

Equilibrating the gel in transfer buffer removes salts that may increase conductivity and heat during transfer. Be careful to equilibrate for the recommended time, as longer equilibration can result in protein diffusion.

  1. After electrophoresis, remove the gel from the cassette.

  2. Place the gel in a shallow tray containing 100 ml (for Midi Gels and E-PAGE™ Gels) or 50 ml (for Mini Gels) of the appropriate Transfer Buffer. Equilibrate for 10 minutes on an orbital shaker.


E-PAGE™ gels: Remove the gel from the transfer buffer and gently rub a gloved finger over the well side of the gel to remove small gel pieces from the gel surface. Re-submerge the gel in transfer buffer to rinse away any gel pieces adhering to the gel, as they can cause air bubbles and field distortion during transfer.
 
Nitrocellulose or Nylon

  1. Use a pre-cut membrane or cut a membrane to the appropriate size for your gel.

  2. Soak the membrane in the appropriate Transfer Buffer for a few minutes in a shallow tray.


PVDF

  1. Use a pre-cut Invitrolon™/Filter Paper Sandwich or cut a PVDF membrane to the appropriate size for your gel.

  2. Pre-wet the membrane for 30 seconds in methanol, ethanol, or isopropanol. Briefly rinse the membrane in deionized water.

  3. Soak the membrane in the appropriate Transfer Buffer for a few minutes in a shallow tray.


 
Gel Layout

We recommend using the following gel layouts in the blotter, to ensure even pressure of the plates on the gel stack. 
                                                                                                                  
                                   
Compression of the Gel Stack

If you are blotting only one Mini Gel, the weight of the cathode plate lid alone will apply enough pressure to maintain good gel/membrane contact. If you are blotting Midi Gels or two or more Mini Gels, it is necessary to slightly tighten the knobs on the Novex® Semi-Dry Blotter to ensure even pressure of the plates on the gel stack(s). Tighten the knobs as described in the following protocol.
It is important that the pressure on the gel stack(s) be even without being too firm. Tighten the knobs roughly evenly with regard to torque, not necessarily with regard to rotational distance. Too little compression can allow proteins to migrate between the gel and membrane, causing protein band smearing. Too much compression can distort the gel.
You can apply a small piece of tape to one of the bumps on each knob to make it easier to determine the rotational distance.
 
Semi-Dry Blotting Protocol

Follow the instructions below to blot 1–2 Midi Gels or 1–4 Mini Gels using the Novex® Semi-Dry Blotter:

  1. In a shallow tray, briefly soak 2 stacked pieces of 2.5-mm thick Blotting Filter Paper in the appropriate Transfer Buffer. Several pieces of thinner blotting paper can be used to produce a stack of equivalent thickness.

  2. Remove any air bubbles trapped between the filter paper sheets by rolling the stack with a blotting roller while it is still submerged in buffer. Note:  Removing air bubbles is essential, as they can block the transfer of biomolecules.

  3. Place the stack of pre-soaked Blotting Filter Paper on the anode plate of the Novex® Semi-Dry Blotter. Remove any air bubbles between the paper and plate by rolling the stack with the blotting roller.

  4. Place the pre-soaked blotting membrane on top of the Blotting Filter Paper stack and remove any air bubbles with the blotting roller.

  5. Carefully remove the gel from the transfer buffer and place on top of the blotting membrane. (E-PAGE™ gels: Place the flat side of the gel on the blotting membrane such that the well side of the gel is facing up.) Gently remove any air bubbles with the blotting roller or a wet gloved finger. Note:   Be careful not to disturb the gel after it has been placed on the membrane. Moving the gel can result in protein smearing on the membrane. Optional:  To physically support the gel, making it easier to pick up and move, place a piece of thin filter paper (included with the membrane/filter paper sandwiches from Invitrogen) on top of the gel in the transfer buffer. Do not fully submerge the paper in buffer; a small amount of buffer on top of the gel will allow the paper to lightly adhere to the gel surface. Then carefully slide a gel knife under one corner of the gel and lift the gel/filter paper out of the tray. After the gel has been placed on the blotting membrane, gently peel away the thin filter paper.

  6. E-PAGE™ Gels only: Fill the wells of the gel with Transfer Buffer after it has been placed on the stack. Soak the E-PAGE™ Blotting Pad in Transfer Buffer, forcing air out of the pad by pressing it with a gloved finger, and then place E-PAGE™ Blotting Pad on the gel and gently roll out air bubbles.

  7. Briefly soak the remaining 2 stacked pieces of 2.5-mm thick Blotting Filter Paper in the appropriate Transfer Buffer. Remove any air bubbles with the blotting roller while the stack is still submerged in buffer.

  8. Place the stack of pre-soaked Blotting Filter Paper on the gel. Ensure that the filter paper sheets are aligned properly and flush with the gel/membrane sandwich. Gently remove any air bubbles with the blotting roller.

  9. Repeat Steps 1–8 for each gel you are blotting. When finished, make sure there are no puddles of buffer on the anode plate.

  10. Place the cathode plate lid on the stack, taking care to properly align the lid holes with the screws and the anode plate electrical connector. Be careful not to disturb the blot sandwich.

  11. If you are blotting only one Mini Gel, the weight of the cathode plate lid alone will apply enough pressure to maintain good gel/membrane contact. For two or more Mini Gels, or if you are blotting Midi Gels or E-PAGE™ gels, thread the knobs onto the blotter screws and spin them down until they just make contact with the lid. Then tighten the knobs another ½ to 1 turn, depending on the number of gels in the blotter (the greater the number of gels, the more tightening required). Note:   Tighten the knobs roughly evenly with regard to torque, not necessarily with regard to the number of turns. Do not overtighten.

  12. Connect the power cables from the blotter to the power supply, making sure that the red-to-red and black-to-black connections are correct.

  13. Mini or Midi Gels: Transfer at 20 V for 30–60 minutes. E-PAGE™ Gels: Transfer at 25 V for 30–60 minutes. (The transfer time may vary depending on the properties of the proteins, and should be determined empirically.)

  14. Proceed to Post-Transfer
TOP

Post-Transfer

Post Transfer Disassembly and Cleaning

To disassemble the blotter and gel stack:

  1. Remove the knobs and lift off the lid of the Novex® Semi-Dry Blotter.

  2. Carefully separate the membrane from the blotting stack and proceed with post-transfer analysis (see below). The gel may be retained for post-transfer staining.

  3. Rinse the Novex® Semi-Dry Blotter with deionized water and air dry after each use.

For repairs and service, contact Technical Service.
 
Post Transfer Analysis

After transfer, you may proceed to immunodetection, store the membrane for future use, or stain the membrane.

  • For immunodetection of proteins, use the WesternBreeze® Chromogenic or Chemiluminescent Immunodetection Kits available from Invitrogen or any other immunodetection kit.
  • For storing nitrocellulose membranes, air dry the membrane and store the membrane in an air-tight plastic bag or dish at room temperature or 4ºC. Avoid storing nitrocellulose below -20ºC, as it may turn brittle.
  • For storing PVDF membranes, air dry the membrane and store in an air-tight plastic bag or dish at room temperature, 4ºC, or -80ºC. When you are ready to use the membrane, re-wet the membrane with methanol for a few seconds, followed by thorough rinsing of the membrane with deionized water to remove methanol.
  • For staining membranes after blotting, you may use any total protein membrane staining methods such as Coomassie® Blue R-250, Ponceau S, Amido Black, SYPRO® Ruby Protein Blot Stain, or SYPRO® Rose Plus Protein Blot Stain. You may also use SimplyBlue™ SafeStain with dry PVDF membranes; to avoid high background, do not use SimplyBlue™ SafeStain on nitrocellulose membranes.
  • If you do not detect any proteins on the membrane after immunodetection or staining, refer to Troubleshooting. Refer to the immunodetection kit manufacturer’s recommendations for optimizing immunodetection.
TOP

Troubleshooting

Review the information below to troubleshoot your experiments using the Novex® Semi-Dry Blotter.

Problem
Cause
Solution
Transfer efficiency is poor
Voltage is too low
 
1-mm thick polyacrylamide gels (mini and Midi Gels) should be transferred at 20 V, E-PAGE Gels at 25 V (approximately 15 V/cm field strength).
 
 
Power supply is inappropriate for semi-dry transfer
Some power supplies will shut off or blow a fuse when run at the conditions required for semi-dry transfer. Semi-dry transfer requires low voltage (20 V) and high current. Check with the manufacturer of the power supply to determine whether it is appropriate for semi-dry transfer.
 
Transfer performed for too short a time
Increase the amount of time for transfer. Typical transfer times range from 30 to 60 minutes.
 
Transfer sandwich was assembled in the wrong order
The Novex® Semi-dry Blotter is configured with the cathode on the top, and anode on the bottom. This results in a downward transfer of proteins from the gel onto the membrane. Follow the instructions carefully when assembling the transfer sandwich.
 
The pH of the transfer buffer is too close to the isoelectric point of the protein
The transfer buffers should be at the optimal pH if prepared as described in this manual. Do not adjust the pH with acid or base as this will increase the conductivity of the buffer and result in higher current during transfer.
 
Too much methanol in the transfer buffer
Reducing the amount of methanol can help elute proteins from the gel, but can reduce binding to nitrocellulose membranes.
 
High-percentage gels restrict transfer
Higher percentage acrylamide or crosslinkers can restrict elution of proteins. Use the lowest percentage acrylamide possible to separate your proteins.
 
Puddles of buffer were present on the plates, allowing the current to bypass the stack
Always clean up the lower plate before closing the lid of the transfer apparatus. Do not squeeze the stack excessively, as this removes transfer buffer from the blotting paper and also creates puddles that the current can pass through.
All membranes
Insufficient binding of proteins to the membrane
Air spaces are interfering with contact between the gel and the membrane
Roll the membrane with a blotting roller (or a clean test tube or pipet) before placing the gel on the membrane, then remove any air bubbles between the gel and membrane with a blotting roller or a wet gloved finger. Transfer will not occur where the gel is not in contact with the membrane.
 
Electrophoretic conditions were incorrect or not ideal
Running conditions, sample preparation, percentage acrylamide, and many other variables can affect the migration and resolution of proteins. Please review your electrophoresis conditions.
 
Under- or over-compression of gel
Follow the compression guidelines in this manual. Too little compression can allow proteins to migrate between the gel and membrane, causing protein band smearing. Too much compression can distort the gel.
Nitrocellulose membrane
Insufficient binding of proteins to the membrane
Over-transfer through the membrane
Use 0.2-µM pore size nitrocellulose instead of 0.45 µM, or use PVDF with a higher binding capacity.
 
Not enough methanol in the transfer buffer
Increase the concentration of methanol.
 
Low molecular-weight proteins are not binding well or are being washed away
Use glutaraldehyde to crosslink the proteins to the membrane and use Tween-20 in the wash steps.
 
SDS is included in the transfer buffer
Do not use SDS in the transfer buffer.
PVDF membrane
 
Smeared or swirled transfer and missing bands
Membrane was dried out before it was added to the transfer sandwich
Membrane should be completely gray and slightly translucent when added to the sandwich. If it has dried out, rewet in methanol and equilibrate in transfer buffer.
 
 
Alcohol was not used to prewet the membrane
PVDF is hydrophobic and requires a short soak in methanol or ethanol prior to transfer.
 
Brown coloration of membrane or cracking of gel after transfer.
Membrane was not thoroughly wetted.
Always pre-wet the membrane according to the manufacturer’s instructions. White spots indicate dry areas of the membrane.
 
 
Too much current
Use a low conductivity transfer buffer such as those recommended in this manual.
 
TOP
LT062