Posts Tagged ‘western blotting’

Revolutionizing protein research

 :: Posted by American Biotechnologist on 06-19-2012

Western blotting, the backbone of protein research, is a universal lab procedure. While the premise of a western blot is simple — target proteins are identified and quantitated via antibody-antigen interactions — the traditional workflow is labor intensive and time consuming. Researchers have long sought a faster solution — an archetype that would streamline the entire process of separation, transfer, and visualization of results without compromising data quality. Has anybody been successful at this endeavor? Yes, a chosen few have! Want to become part of this exclusive club?

Find out more about a revolutionary new workflow that will help you cut your western blotting time in half!

Keeping it constant: A lesson in protein transfer

 :: Posted by American Biotechnologist on 05-22-2012

Power supplies that are used for electrophoresis hold one parameter constant (either voltage, current, or power). The PowerPac™ HC and PowerPac Universal power supplies also have an automatic crossover capability that allows the power supply to switch over to a variable parameter if a set output limit is reached. This helps prevent damage to the transfer cell.
During transfer, if the resistance in the system decreases as a result of Joule heating, the consequences are different and depend on which parameter is held constant.

Transfers Under Constant Voltage
If the voltage is held constant throughout a transfer, the current in most transfer systems increases as the resistance drops due to heating (the exception is most semi-dry systems, where current actually drops as a result of buffer depletion). Therefore, the overall power increases during transfer, and more heating occurs. Despite the increased risk of heating, a constant voltage ensures that field strength remains constant, providing the most efficient transfer possible for tank blotting methods. Use of the cooling elements available with the various tank blotting systems should prevent problems with heating.

Transfers Under Constant Current
If the current is held constant during a run, a decrease in resistance results in a decrease in voltage and power over time. Though heating is minimized, proteins are transferred more slowly due to decreased field strength.

Transfers Under Constant Power
If the power is held constant during a transfer, changes in resistance result in increases in current, but to a lesser degree than when voltage is held constant. Constant power is an alternative to constant current for regulating heat production during transfer.

The above information was adapted from Bio-Rad’s protein blotting guide. For more great information, be sure to download the Protein Blotting Guide from Bio-Rad Laboratories.

Top 8 reasons for high background noise on your western blot

 :: Posted by American Biotechnologist on 05-02-2012

The following are the top 8 causes for overall high background on your western blot and potential solutions:

  1. Blocking was incomplete
    • Increase the concentration of blocker
    • Increase the duration of the blocking step
    • Use a different blocking agent
  2. Blocker was impure
    • Use a pure protein such as BSA or casein as a blocker
  3. Wash protocols were insufficient
    • Increase the number, duration, or stringency of the washes
    • Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween 20
    • Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding
  4. The blot was left in the enzyme substrate too long (colorimetric detection)
    • Remove the blot from the substrate solution when the signal-to-noise level is acceptable, and immerse in diH2O
  5. Contamination occurred during electrophoresis or transfer
    • Discard and prepare fresh gels and transfer solutions
    • Replace or thoroughly clean contaminated foam pads if a tank blotter was used
  6. Excessive amounts of protein were loadedon the gel or too much SDS was used inthe transfer buffer. Proteins can pass through the membrane without binding and recirculate through a tank blotting system.
    • Reduce the amount of protein on the gel or SDS in the transfer buffer
    • Add a second sheet of membrane to bind excess protein
  7. The primary or secondary antibody was too concentrated
    • Increase antibody dilutions
    • Perform a dot-blot experiment to optimize working antibody concentration
  8. Incubation trays were contaminated
    • Clean the trays or use disposable trays

For more great information, download the protein blotting guide from Bio-Rad Laboratories.

Cut Western Blot Time in Half with V3

 :: Posted by American Biotechnologist on 04-16-2012

How anxious are you to get experimental results? Don’t you wish that you could cut those 2 day western blots in half? If so, the V3 workflow method by Bio-Rad Laboratories will surely get you excited.

V3, which stands for visualizing protein separation, verifing protein transfer, and confidently validating blot data via total protein normalization, uses a 5-step process to cut traditional western blotting times down from 2 days to one. The steps include:

  1. 15 minute protein separation
  2. stain free band imaging
  3. three minute protein transfer
  4. instant visualization of protein transfer onto membrane
  5. stain-free total protein normalization for faster quantitative results

For more information visit www.bio-rad.com/ad/V3pr.

Transfering high molecular weight proteins

 :: Posted by American Biotechnologist on 02-20-2012

We are all intimately familiar with protein blotting techniques which have been a cornerstone of the biochemicstry/biology lab for the past 30 years.

As is well known, the efficiency of protein migration is affected by various factors including the size and charge of the protein, and protocol optimization is often needed on a protein-specific basis. In fact, it can be particularly challenging to transfer large molecular weight proteins alongside small molecular weight proteins, as transfer conditions may cause small proteins to blow through the membrane.

Currently there are three popular techniques for protein transfer: the tank transfer, the semi-dry blotting method and the fast blotting “turbo” technique (for transfer within 3-10 minutes).

In the attached paper, Transfer of high molecular weight proteins to membranes: a comparison of transfer efficiency between blotting systems, Bio-Rad Laboratories presents a comparison of the various blotting techniques across a wide range of molecular weights with a particular emphasis on large proteins (more than 200kD).