Improper sample preparation can cause headaches when preparing lysates:
- Work quickly
- Keep the low temperature environment
- Increase protease
- Packing to avoid repeated freezing and thawing
If your lysate contains a lot of DNA, it will become sticky. A large amount of DNA makes the sample difficult to pipette and may cause streaks or smudges on the gel. If this is the case, be sure to add DNase to the lysate before adding the loading buffer.
For denatured gels, don't forget to heat the sample at 98 ° C for 5 minutes before electrophoresis. This will completely denature the sample and ensure a clean, sharp strip.
Some tips are available for reference:
Using the filtered buffer, carefully check the buffer before electrophoresis to ensure that the buffer does not precipitate over time.
When removing the gel comb, do so very slowly to avoid collapse or distortion of the hole.
Use the sampler to load. When loading, be sure to place the tip as close as possible to the bottom of the well, slowly adding the sample while carefully removing the sampler.
Load the same volume in each well, not empty.
Low voltage jogging, especially when initially concentrating the sample.
· After electrophoresis is complete, use a clean blade to remove the gel and bottom gel. These can result in slight height differences that can interfere with the transfer.
Transfer
Transfer is the process of transferring proteins from a polyacrylamide gel to a membrane.
- Make sure all materials of the “sandwich†are the same size. Filter paper, film and gel should all be the perfect choice. This will prevent nasty bubbles from appearing on your membrane.
- Do not stretch the gel when removing the gel from the rubber sheet. After removing the bottom and the hole from the gel with a blade, place a few pieces of pre-moistened filter paper on the gel and gently flatten it. The filter paper will act as a support and carefully peel off the gel.
There are many things that can go wrong here.
Choose the appropriate standardization strategy. Let's face it, no matter how careful you are, you may get a defective gel or film, which will ruin your day's experiment. However, if you use total protein staining or load a control antibody to normalize your data, you can save your experiment. For example, using AzureRed total protein stain, compatible with downstream western blot experiments, and sapphire dual-mode multi-spectral imaging system, multiple fluorescence detection can be performed, which is more accurate and more convenient to use.
Make sure the target and internal parameters have the same linear range. Chemiluminescence is semi-quantitative imaging, and it is especially important to avoid saturation.
Choose a high quality primary antibody that has been fully validated and cited in the literature.
Use a suitable substrate to avoid signal saturation. Azure offers Radiance and Radiance plus chemiluminescent substrates to meet all your sensitivity needs.
Consider the appropriate background removal method to accurately quantify the bands.
eRed total protein stain, compatible with downstream western blot experiments, combined with sapphire dual-mode multi-spectral imaging system, can perform multiple fluorescence detection, more accurate and more convenient to use.
Make sure the target and internal parameters have the same linear range. Chemiluminescence is semi-quantitative imaging, and it is especially important to avoid saturation.
Choose a high quality primary antibody that has been fully validated and cited in the literature.
Use a suitable substrate to avoid signal saturation. Azure offers Radiance and Radiance plus chemiluminescent substrates to meet all your sensitivity needs.
Consider the appropriate background removal method to accurately quantify the bands.
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