Protein concentration in the analyzed sample should be estimated before the sample is loaded on a gelSurprisingly, many laboratories do not have the crucial step of analyzing the protein concentration included in their protocol. Protein is often extracted from a range of samples, and loaded on the gel based on volume, rather than protein concentration. This would work if each sample was extracted with exactly the same efficiency, but this is not the case; especially when protein extraction is done manually. Accurate protein quantification is crucial in securing reproducible and reliable data when doing Western blot analyses.Some assays used to measure protein concentration are:
The following parameters should be considered before choosing a specific assay:
| There is a wide range of assays, available from different suppliers, which are compatible with components commonly used in extraction buffers, like detergents, urea, TRIS and HEPES. In case of photosynthetic tissues, loading can be done based on chlorophyll content, which can be easily estimated following the extraction. Loading/protein or chlorophyll concentration is highly recommended! |
Latest
What parameters should be consider when transferring HMW proteins?2024-03-01 Why some antibodies have a broad species reactivity, while the others do not?
2024-02-20 Are directly conjugated primary antibodies always the best choice?
2023-12-28 How to obtain sharper protein bands in Western blot?
2023-12-20 Are monoclonal antibodies better, compared to polyclonal antibodies?
2023-11-30 Why, to detect some proteins, 6M urea must be included in the extraction and loading buffers?
2023-09-01 I have not used this antibody before, what conditions of Western blot do you recommend?
2023-07-27 Which subclasses of immunoglobulin G are found in rabbit serum?
2023-07-18 Antibody suddenly does not work any longer, what can be the reason?
2023-07-11 Can an antibody made to a full-length protein distinguish between 2 isoforms?
2023-05-10
Archive
- March - 2024
- February - 2024
- December - 2023
- November - 2023
- September - 2023
- July - 2023
- May - 2023
- March - 2023
- January - 2023
- December - 2022
- November - 2022
- October - 2022
- September - 2022
- August - 2022
- June - 2022
- May - 2022
- March - 2022
- February - 2022
- January - 2022
- November - 2021
- October - 2021
- August - 2021
- June - 2021
- May - 2021
- April - 2021
- March - 2021
- February - 2021
- January - 2021
- December - 2020
- November - 2020
- October - 2020
- September - 2020
- August - 2020
- July - 2020
- June - 2020
- May - 2020
- April - 2020
- March - 2020
- January - 2020
- November - 2019
- October - 2019
- March - 2019
- April - 2017
- February - 2017
- May - 2016
- February - 2014
- September - 2013
- December - 2010