ELISAs are more suitable for this purpose and generally more sensitive. Since the signal produced is not linear, it should not be used to attach a precise concentration to a particular sample. The reason for this is two-fold: first, there will be variations in loading and transfer rates between the samples in separate lanes and on separate blots that will need to be normalized before a more precise comparison can be made second, the signal generated will not be linear across the concentration range of samples due to substrate availability and linear responsiveness of the detection method. That is, it provides a relative comparison of protein levels, but not an absolute measure of quantity for a specific target protein in a particular experiment. It is very important to be aware that the data produced with a Western blot is typically considered to be semi-quantitative. If there are no bands on the blot, or if there are unexplained blotches or uneven signal, troubleshooting advice is offered in Chapter 6. The focus of this chapter is analysis and as such the data itself will be discussed along with examples of different types of Western blot data from research and clinical settings to demonstrate the flexibility of the technique. In some cases the data may be more complex, showing unexpected sizes, multiple bands, or alteration in bands following a particular treatment. Their identity is confirmed by comparison to molecular weight markers (for size) and a positive control (size and signal). In the majority of cases, bands corresponding to the target protein will become visible upon treatment of the blot with substrate. Nitrocellulose can be blocked easily and will provide a good signal-to-noise ratio.The data produced with a Western blot is usually quite easy to interpret. It is resilient and stable and better for protein retention. PVDF is a better choice if you plan to strip and re-probe your blot. It is worth some experimentation with membrane types to identify the membrane that will provide you with the optimal 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.īiorbyt can guide you to the perfect choice to fit your protocol. 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. Variations in concentration will produce variations in result, it is worth the effort before-hand to obtain clear and accurate results. Examples are HRP-labeled secondaries used for chemiluminescent detection. In some cases, titration of secondary antibodies is also indicated. 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. Biorbyt recommends that antibody titration is carried out each time your conditions are changed. You can experiment with a range of antibody concentrations by varying antibody dilution. Concentration of antibody to antigen, pH and temperature are some of the factors that affect the rate of binding. Select Western blot validated primary antibody specific to your protein of interest.Ĭhoose the correct secondary antibody for detection.īiorbyt are happy to advise you on antibody selection and our secondary antibody selection tool is here to help you.Įstablish the optimum antibody concentration.
0 Comments
Leave a Reply. |