General Western Blotting and Detection Procedures

Regardless of the type of transfer used, the general setup for an electrophoretic western transfer is similar. Specific methods for setting up wet or semi-wet, semi-dry, and dry transfers are shown in the tables and figures that follow. Additionally, please view the videos (at the links listed later in this section) for instruction on how to set up an electrophoretic gel transfer.

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In brief, the transfer consists of a gel and a membrane sandwich, with the gel placed closer to the cathode and the membrane positioned closer to the anode. When current is applied, the negative charge of the proteins (conferred by the SDS applied during electrophoresis) causes them to migrate from the gel onto the membrane. The transfer is conducted under constant voltage, with the time and current settings inversely related (i.e., higher current decreases transfer time). It is important to note that higher currents may overheat the system, resulting in melting of the gel or poor transfer.

Setting Up a Wet or Semi-Wet Transfer

 

setup of a blot for a wet/semi-wet transfer.
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Figure 1. A wet/semi-wet transfer setup for western blotting.
 

 

Table 1, below, shows the recommended settings for transferring proteins in Life Technologies gels using the XCell II™ semi-wet blot module. Note that 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. Figure on left shows the correct setup of a blot for a wet/semi-wet transfer.

Recommended wet transfer conditions for performing western blotting with various Life Technologies gel types

Gel Transfer Buffer Membrane Power Conditions
NuPAGE® Novex® Bis-Tris gel1X NuPAGE® Transfer Buffer with
10% methanol
0.1% NuPAGE® Antioxidant for
reduced samples
Nitrocellulose or PVDF

30 V constant for 1 hr
Expected current
Start: 170 mA
End: 110 mA

NuPAGE® Novex® Tris-acetate gel1X NuPAGE® Transfer Buffer with
10% methanol
0.1% NuPAGE® Antioxidant for
reduced samples
Nitrocellulose or PVDF

30 V constant for 1 hr
Expected current
Start: 220 mA
End: 180 mA

Tris-glycine gel
Tricine gel
1X Tris-glycine transfer buffer with 20% methanolNitrocellulose or PVDF25 V constant for 1–2 hr
Expected current
Start: 100 mA
IEF gel

1X Tris-glycine transfer buffer with 20% methanol

 

0.7% acetic acid, pH 3.0

Nitrocellulose or PVDF

 

Nitrocellulose or PVDF

25 V constant for 1 hr
Expected current
Start: 65-85 mA

10 V constant for 1 hr
Expected current
Start: 65-85 mA

TBE gel
0.5X TBE running bufferNylon

30 V constant for 1 hr
Expected current
Start: 39 mA
End: 35 mA

TBE-urea gel0.5X TBE running bufferNylon

30 V constant for 1 hr
Expected current
Start: 39 mA
End: 35 mA

DNA retardation gel0.5X TBE running bufferNylon

30 V constant for 1 hr
Expected current
Start: 39 mA
End: 35 mA

 

Table 1

Setting Up a Semi-Dry Transfer

Table 2 contains the recommended settings for transferring proteins using the Novex® Semi-Dry Blotter. The figure belows shows the correct setup of a blot for a semi-dry transfer. It is important that the pressure on the gel stack(s) be even without being too firm.

Gel type Transfer buffer Membrane Power conditions
NuPAGE® Novex® Bis-Tris and Tris-acetate gels2X NuPAGE® Transfer Buffer with 10% methanol and 0.1% NuPAGE® Antioxidant for reduced samplesNitrocellulose or PVDF20 V constant for 30–60 min
E-PAGE™ gel2X NuPAGE® Transfer Buffer with 0.1% NuPAGE® Antioxidant for reduced samplesNitrocellulose or PVDF25 V constant for 30–60 min
Tris-glycine gel Tricine gel2X Tris-glycine transfer buffer with 20% methanolNitrocellulose or PVDF20 V constant for 30–60 min

Table 2


 

 

semi-dry-transfer setup
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Figure 2.  A semi-dry transfer setup for western blotting.

Setting Up a Dry Transfer with iBlot® Gel

Table 3 contains the recommended settings for transferring proteins in Life Technologies gels using the iBlot® Gel Transfer Device. Figure 4 shows the correct setup of a blot for a dry transfer using the iBlot® Gel Transfer Device.

Gel type Program Voltage Run time
E-PAGE™ 48 gelP223 V7–8 min
E-PAGE™ 96 gelP223 V7–8 min
Novex® midi gel, 1 mm thickP223 V6 min
2 mini gels (1.0 or 1.5 mm thick)P223 V6 min
T1 mini gel (1.0 or 1.5 mm thick) using mini transfer stacksP223 V6 min

 

Table 3

 

 

dry blot setup
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Figure 3.  The setup of a blot for a dry transfer with the iBlot® Gel Transfer Device.

Probing and Detection

Once proteins have been transferred onto a membrane, they can be probed with antibodies. The proteins on the membrane are exposed to antibodies either through passive diffusion or through electrically driven binding. Passive diffusion involves submerging the membrane in a solution that contains the diluted antibody, washing of the membrane, and then incubation with a diluted secondary antibody. The extent of dilution that works best will depend on the antibodies used. Electrically driven probing of a membrane is performed using the iBlot® Gel Transfer Device and the iBlot® Western Detection Kits, which have anode and cathode stacks designed to move antibodies toward the transfer membrane for western detection.

After the membrane has been probed with labeled antibodies, the target proteins on the membrane can be detected by the method appropriate to the label. Enzymatic detection involves the addition of an enzyme substrate to the membrane followed by washing unbound reaction products and unreacted substrate away from the membrane. Chromogenic reaction products can be visualized by examining the membrane directly or by analyzing a visible-light image of the membrane. Chemiluminescent reaction products can be detected by exposing film to the membrane or by using a chemiluminescence scanner. Fluorescently labeled antibodies can be visualized directly by placing the membrane in an appropriate scanning instrument that can excite
and detect the bound fluorophore.

Stripping and Reprobing Membranes

When multiple proteins need to be detected on a single membrane, the traditional approach is to strip the primary and secondary antibodies from the blot and reprobe with different primary and secondary antibodies. One common method of antibody removal is the use of a heated, low-pH solution containing glycine. The combination of heat and low pH causes dissociation of the antibody–antigen complex. After the membrane has been stripped, it can be reprobed with different antibodies. When planning to strip and reprobe a membrane, it is recommended that you use PVDF for the transfer. Because PVDF is a more durable material, it is less prone to damage during the stripping process than is nitrocellulose.