i extracted total protein of hela cell culture via RIPA lysis buffer, or directly lysed cells with 1x SDS sample buffer. then did 12% SDS-PAGE with proper procedure and antibody, both yielded only 1 band, which seems like LC3B-ii.
i wanna have both LC3B-I and LC3B-II bands. how can i modify the assay?
Hi there.
There are several reasons for that .
Firstly, can be the gel
Or early response of cells , so yielding LC3B ii very early than expected.
Try to checkout transfer process.
Hello Hongyu Chen
In a Western blot, the LC3B-I band is typically observed at a higher molecular weight, around 16-18 kDa, while the LC3B-II band, the lipidated form, is seen at a lower molecular weight, around 14-16 kDa. Are you sure the band you obtained is LC3BII? Because the molecular weights of both LC3B-I and LC3B-II are very close to each other.
Nevertheless, there are a few modifications that I may want to suggest.
1. You should try to reduce the severity of your lysis step by using less harsh lysis conditions, such as a non-ionic detergent buffer will help to prevent LC3B-II from being further degraded. Instead of RIPA buffer, which contains ionic detergents like SDS, use a buffer with non-ionic detergents (like NP-40 or Triton X-100). RIPA buffer's ionic detergents can be too harsh, leading to complete degradation of LC3B-II. There is a possibility that harsh lysis conditions can strip away the lipidated form of LC3B (LC3B-II), causing it to become fully delipidated and appear as a single band at the LC3-I position.
2. In conditions of high autophagic flux, LC3B-II may be rapidly degraded, leading to a low or non-existent band. So, to prevent this degradation, you may treat your cells with a lysosomal inhibitor like bafilomycin A1 before harvesting.
3. Try reducing the heating time as over-heating your sample can cause further degradation.
4. A standard 12% SDS-PAGE gel may not provide enough resolution to separate the LC3B-I and LC3B-II bands. Switch to a 15% or 16% gel, or a 4–20% gradient gel, to improve separation.
5. You may need to use a lower voltage and run the gel for a longer time to achieve better resolution. Inappropriate running conditions, such as high voltage, can cause the bands to overlap.
Best,
To successfully visualize both LC3B-I and LC3B-II bands, you need to modify your protocol to preserve the labile LC3B-I form and prevent its post-lysis conversion or degradation. The key is to use a gentler, specially formulated lysis buffer that replaces the standard RIPA buffer. This new buffer should contain a critical additive, N-Ethylmaleimide (NEM), which is a cysteine protease inhibitor that blocks the ATG4B enzyme responsible for de-lipidating LC3B-II back to LC3B-I after the cells are lysed. The lysis procedure itself must be performed with speed and kept ice-cold throughout, from rinsing the cells with cold PBS to scraping them directly into the cold lysis buffer containing protease inhibitors and NEM. After a centrifugation step to clear the lysate, the protein sample should be mixed with SDS loading buffer and then boiled immediately before loading onto the gel. Finally, including experimental controls, such as treating cells with Bafilomycin A1 to block autophagy and cause LC3B-II accumulation, is essential for confidently interpreting your western blot results and confirming the identity of the bands.
briefly, the important points are: new gentler ice-cold lysis buffer with PIC and NEM, fast and cold lysis, clearing the lysate by centrifugation, and controlled boiling.
- Badges
- Science topic