Posts Tagged ‘western blotting’

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.

Protein blotting guide for novice and advanced users

 :: Posted by American Biotechnologist on 11-15-2011

Protein blotting is a staple technique of most molecular biology and proteomics laboratories. In previous posts, we discussed topics such as semi-dry protein transfer and protein transfer methods, and we even did a multi-part series on western blotting.

Now, we are proud to present you with a 43 page protein blotting guide put together by Bio-Rad Laboratories. The guide is organized into two parts which cover the theory and methods behind protein blotting. You will learn topics such as methods and instrumentation, the difference between various membranes and tranfer buffers, the ins and outs of transfer conditions, detection and imaging and a host of different blotting and detection protocols.

The guide is fairly technical and is appropriate for both novice and advanced users alike.

Click on the link to download the Protein Blotting Guide now.

An inside look at Bio-Rad product development

 :: Posted by American Biotechnologist on 10-24-2011

The development of Bio-Rad’s award winning Gel Doc™ EZ system represents the culmination of years of progress toward simplifying the imaging step of gel electrophoresis workflows. As with all new product releases, launching the Gel Doc EZ system has required the vision and expertise of a cohesive internal team as well as testing and feedback from the scientific community. The key players involved in bringing the Gel Doc EZ system to market describe, in their own words, the critical steps — from conception to validation to product launch — involved in development of this latest imaging innovation.

Click here to read what the experts say goes into product development.

Renee LeMaire-Adkins, Suresh Mehta, Keith Kotchou, Nik Chmiel and Kevin McDonald share their thoughts on product develpment

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.