Western Blot Tips and Tricks

Western Blot Tips and Tricks

What is Western Blot?

If you need to identify specific amino-acid sequences from a sample, Western Blotting is the technique you should choose. Target proteins are marked using carefully selected primary and secondary antibodies that enable you to visualize the specific protein you are interested in. Western Blotting, like any other laboratory technique, needs to be carried out with precision and understanding at each stage to achieve the best results. With years of experience behind us, we have learned what works best. Biorbyt aim to provide guidance to save you time and frustration and make the process easier and more accurate optimizing your results.

With Western blot, you are aiming for the clearest results via an increased signal-to-noise ratio; displaying clearly the specific bands and minimizing the presence of non-specific bands. There are many steps that can be taken to achieve that goal.

Western blotting protocol in a nutshell:

  1. Extraction of protein using cell lysis
  2. Separation of molecules according to size/weight using electrophoresis
  3. Transfer molecules to solid membrane
  4. Incubation with primary and secondary antibodies to visualise target protein by marking
  5. Washing off unbound antibodies
  6. Detection of protein of interest

See our recommended Western blot protocol. We also have the following tips to save you time and effort in the process:

Below are the most basic validation steps that should have been carried out for your antibody:

Antibody Selection

  • Select Western blot validated primary antibody specific to your protein of interest.

  • Choose the correct secondary antibody for detection.

  • Biorbyt are happy to advise you on antibody selection and our secondary antibody selection tool is here to help you.

  • Establish the optimum antibody concentration. Concentration of antibody to antigen, pH and temperature are some of the factors that affect the rate of binding. You can experiment with a range of antibody concentrations by varying antibody dilution. Biorbyt recommends that antibody titration is carried out each time your conditions are changed. Our data sheets provide a dilution factor range as a guide and we recommend you start with a concentration close to the middle of the range, titrating up or down as required.

  • In some cases, titration of secondary antibodies is also indicated. Examples are HRP-labeled secondaries used for chemiluminescent detection. Variations in concentration will produce variations in result, it is worth the effort before-hand to obtain clear and accurate results.

Protein Extraction Tips

  • During extraction of protein from tissue or cells keep tissue culture dish cooled to a temperature of -4°C

  • After centrifuging the mixture you will need to add lysis buffer and protease inhibitor cocktail. You may need to repeat this process with differing concentrations of protease inhibitor cocktail to obtain a high enough protein concentration

Sample Preparation

  • Use a Bradford, Lowry or BCS assay to establish protein concentration then ensure you load a uniform amount of protein

  • Each well should contain approximately 20 – 40 ug of protein extract.

  • Add the same volume of sample buffer to each empty well

  • Ensure the volume in each lane is equalized to a total volume of µL

Preparation of Gel

  • The quality of your gel must be good to get a good transfer to membrane

  • To obtain a uniform gel see our protocol guide for recommended acrylamide percentage

  • For gel solidification, prepare the rack for the 10% stacking gel solution. Biorbyt recommends that you add the gel slowly using a pipette

  • 10% AP or TEMED will cause solidification, therefore only add these reagents at the end of gel preparation

  • Remove excess water with an absorbent towel using capillary action

  • Keep some gel in a test-tube to check its state of solidification

Electrophoresis

  • Once your running buffer is in the electrophorator and is covering the gel, connect the power supply, connectors: red to red and black to black

  • Samples and markers are added to the gel after careful removal of the combs

  • Ensure you check that you select the correct voltage on the power pack for the gel you are using

Electrotransfer

  • The most commonly used membranes are PVDF (polyvinylidene fluoride) and nitrocellulose, both have a range of different versions and your choice will affect your results. Nitrocellulose membranes also have a range of pore sizes on offer, which you can select according to the sizes or protein you’re needing to separate.

  • Biorbyt can guide you to the perfect choice to fit your protocol. It is worth some experimentation with membrane types to identify the membrane that will provide you with the optimal results.

  • PVDF is a better choice if you plan to strip and re-probe your blot. It is resilient and stable and better for protein retention. Nitrocellulose can be blocked easily and will provide a good signal-to-noise ratio.

  • When adding the methanol soaked PDVF membrane to the gel, ensure no excess liquid and that there are no air bubbles

  • Maintain a 4°C temperature by keeping the transfer apparatus on ice

  • The membrane should be in between the gel and the positive electrode

  • Ensure you use the optimal running time for the thickness of your gel

Blocking

  • There are a wide range of blocking buffers available, each of which works differently in different conditions. There will be one that is the best choice for your antibody-antigen.

  • Your blocking buffer will contain endogenous biotin, glycoproteins and enzymes if it is milk-based. These chemicals will affect your signal.

  • Membrane blocking should be carried out for one hour

  • Biorbyt’s blockers are optimised for specific conditions, we are happy to advise you.

Antibody Incubation

  • We would recommend adding your primary antibody in 5% bovine serum albumin then incubating on a shaker for 12 hours at 4°C

Secondary Antibody

  • See protocol for guidance. After adding secondary antibody, incubate for one hour.

  • Follow this step by repeating the washing procedure then use a pipette to completely the memberane with ECL mix. Incubate for one to two minutes

Washing

  • Continue working on a shaker for this stage to continue agitation. Wash at least three times with TBST for five minutes each time.

  • More washing adjustment options: Altering the washing time or volume of liquid, changing detergent strength.

  • A slight increase of Tween-20 concentration is a good way to fix a high background signal

Visualisation

  • We advise you run a few tests using different exposure times to obtain the clearest results possible. A long exposure time may result in the background appearing too strong.

  • Like with all other stages of Western Blotting, there are many choices available, enabling you to fine-tune your experimental design for increased accuracy and clarity of results. Biorbyt supply a wide range of chemiluminescent reagents and alternatives to allow you to find the perfect option. Consider if you need longer-lasting signal for greater reproducibility, we can provide a more sensitive reagent.

  • We would recommend choosing a film that is designed for chemiluminescent detection. Exposure times can also be changed, we recommend including a range of exposure times within your protocol.