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Archive for the ‘Western blot tutorial series’ Category

Three important points about gel equilibration

 :: Posted by American Biotechnologist on 01-19-2012

So you’ve isolated your protein, ran them on a gel and now you’re ready to transfer them to a membrane to begin western blotting. Sounds simple, right? Not so fast. Don’t forget to equilibrate your gel prior to beginning your transfer. Gel equilibration generally involves rinsing the gel in diH2O and soaking it in transfer buffer for approximately 15 min. While it may sound simple, (and it truly is), it is a step that might make the difference between an ugly blot and one that is publication worthy.

Below are some points to consider about gel equilibration:

  1. Gel equilibration removes contaminating electrophoresis buffer salts. If not removed, these salts increase the conductivity of the transfer buffer and the amount of heat generated during transfer.
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  3. Equilibration also allows the gel to adjust to its final size prior to electrophoretic transfer. Gels shrink or swell to various degrees in the transfer buffer depending on the acrylamide percentage and the buffer composition.
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  5. Equilibration is not necessary (i) when the same buffer is used for both electrophoresis and transfer (for example, native gel transfers), or (ii) when using rapid semi-dry transfer systems such as the Trans-Blot® Turbo™ system (consult the user manual for the system you are using).

To learn more tips and tricks, download the Protein Blotting Guide from Bio-Rad Laboartories.

8 critical tips for western blotting analysis

 :: Posted by American Biotechnologist on 01-12-2012

So you think that protein transfer for western blotting is a piece of cake? Consider these important tips before proceeding:

  1. Use only high-quality, analytical grade methanol. Impure methanol can increase transfer buffer conductivity and yield a poor transfer.
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  3. In many cases, ethanol can be substituted for methanol in the transfer buffer with minimal impact on transfer efficiency. Check this using your samples.
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  5. Do not reuse transfer buffer since the buffer will likely lose its ability to maintain a stable pH during transfer.
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  7. Do not dilute transfer buffers below their recommended levels since this decreases their buffering capacity.
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  9. Do not adjust the pH of transfer buffers unless specifically indicated. Adjusting the pH of transfer buffers can result in increased buffer conductivity, manifested by higher initial current output and decreased resistance.
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  11. Increasing SDS in the transfer buffer increases protein transfer from the gel but decreases binding of the protein to nitrocellulose membrane. PVDF membrane can be substituted for nitrocellulose when SDS is used in the transfer buffer.
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  13. Addition of SDS increases the relative current, power, and heating during transfer, and may also affect antigenicity of some proteins.
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  15. Increasing methanol in the transfer buffer decreases protein transfer from the gel and increases binding of the protein to nitrocellulose membrane.

 

To learn more tips and tricks, download the Protein Blotting Guide from Bio-Rad Laboartories.

Transfer Efficiency: Influence of the Gel Structure

 :: Posted by American Biotechnologist on 10-17-2011

With its proprietary transfer buffer, the Trans-Blot Turbo system generates very fast transfer even for high molecular weight proteins. However, as indicated previously, the gel composition, i.e. the acrylamide – bis-acrylamide network density, influences the transfer efficiency. A protein can more easily move out of the gel during the transfer if it is located in a portion of gel that has the widest pore structure. As proteins above 150 kD are always located on the first top part of a gel, the most efficient transfer of those large proteins is achieved when using gradient gel with a concentration of 4% of acrylamide – bis-acrylamide at the top of the gel. The transfer efficiency of proteins from an Any kD homogeneous acrylamide gel and a 4-20% gradient gel is illustrated.

Qualitative transfer efficiency comparison of HMW proteins on homogeneous acrylamide % and gradient gel. Precision Plus Protein™ Unstained standard and E. Coli homogenate (20 μg) were run on both Criterion TGX Any kD Stain-Free and 4-20% gels at 300V for 18 min. The total protein content was detected with the Stain-Free technology using the Gel-Doc EZ imaging system. The gels were then transferred with the Trans-Blot Turbo system with the 7 min preset program using the Trans-Blot Turbo PVDF transfer packs. The total protein content remaining in the gel is detected with the Stain-Free detection, using the same exposure parameters as used with the gels before the transfer for reliable comparison. The content of protein was also detected with the Stain-Free detection on the membrane. Even if most of the proteins are transferred in 7 min, the gradient gel contains less HMW proteins than the homogeneous gel after the transfer.

Protein Transfer: Relationship Between Power Settings and Transfer Times

 :: Posted by American Biotechnologist on 10-16-2011

In theory, increasing the power input and duration of an electrophoretic transfer results in the transfer of more protein out of a gel.

In practice, however, test runs should be used to evaluate transfer efficiency at various field strengths and transfer times for each set of proteins of interest.

The optimum transfer conditions depend on a number of factors, including the size, charge and electrophoretic mobility of the protein, the type of gel and transfer buffer used, and the type of transfer system being used.

In a semi-dry transfer system, the distance between electrodes is determined only by the thickness of the gel-membrane sandwich, and buffering and cooling capacity is limited to the buffer in the filter paper. As a result, the field strength is maximized in semi-dry systems, and the limited buffering and cooling capacity restricts the transfer time. Though power conditions may be varied with the power supply, semi-dry transfers often operate best within a narrow range of settings.

Semi-Dry Protein Transfer Explained

 :: Posted by American Biotechnologist on 10-14-2011

In semi-dry systems, the distance between the electrodes is limited only by the thickness of the gel and membrane sandwich. As a result, high electric field strengths and high-intensity blotting conditions are achieved. Under semi-dry conditions, some small proteins may be driven throughout the membrane in response to the high field strengths. Moreover, because low buffer capacity limits run times, some large proteins may be poorly transferred. Use of a discontinuous buffer system may enhance semi-dry transfer of high molecular weight proteins (>80 kD). As semi-dry transfers require considerably less buffer and are easier to set up than the tank method, laboratories performing large numbers of blots often favour them.

Novel buffer and material formulations have been developed that can be used with higher electric field strengths than those used in typical semi-dry blotting. These conditions yield complete and extremely rapid transfer, with some systems completing transfer in 3 – 10 min. Such rapid blotting systems do not incorporate external cooling mechanisms, so the high power dissipation may generate more heat than other techniques. Rapid blotting systems are intended for extremely rapid transfers where heat-induced protein denaturation will not affect downstream applications.

The Trans-Blot Turbo system performs semi-dry transfers in as little as 3 min. The system uses prepackaged transfer packs containing a prewet membrane (nitrocellulose or polyvinylidene difluoride:PVDF) and filter paper stacks soaked with a proprietary buffer that allows this fast and efficient transfer. The base unit contains an integrated power supply that drives two independent transfer cassettes, allowing transfer of a total of four mini-format or two midi-format gels. As the transfer cassette electrodes are made with robust permanent stainless-steel and platinum-coated titanium, the Trans-Blot Turbo system also has the flexibility to run traditional semi-dry protocols with hand-made buffers and any membrane, as any semi-dry system.

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