Hi Giulio, Very thanks to your reply! I've treated HEK293T cells with 100nM rapamycin for 1 or 5 hours and I can see the obvious conversion of LC3I to LC3II. So I think it can work properly. The antibody I used is from MBL. I just wondering why I can detect it in HEK293T cells but not in Jurkat and Raji cells.
Hi Giulio, Very thanks to your reply! I've treated HEK293T cells with 100nM rapamycin for 1 or 5 hours and I can see the obvious conversion of LC3I to LC3II. So I think it can work properly. The antibody I used is from MBL. I just wondering why I can detect it in HEK293T cells but not in Jurkat and Raji cells.
Hi Jingting For a start, it seems you are not the first to have this problem (see link). Although I have never used these cell lines, I would recommend blocking the late stages of autophagy using bafilomycin A1 (10 nM for 2 hours for example) or chloroquine. If the turnover of LC3 is very rapid in these cells, you might have to block autophagy to accumulate detectable levels of LC3. You can also combine this treatment with an inducer such as rapamycin (personally I use Torin1 at 250 nM for 3 h with good effect) to further increase LC3-II levels. https://www.researchgate.net/post/Difficulty_detecting_LC3_in_Jurkat_cell_lysate
Hi Jingting For a start, it seems you are not the first to have this problem (see link). Although I have never used these cell lines, I would recommend blocking the late stages of autophagy using bafilomycin A1 (10 nM for 2 hours for example) or chloroquine. If the turnover of LC3 is very rapid in these cells, you might have to block autophagy to accumulate detectable levels of LC3. You can also combine this treatment with an inducer such as rapamycin (personally I use Torin1 at 250 nM for 3 h with good effect) to further increase LC3-II levels. https://www.researchgate.net/post/Difficulty_detecting_LC3_in_Jurkat_cell_lysate
Hello, As first thing I would verify how good is your antibody by treating with Rapamycin or others autophagy inducers to check if you can detect the conversion from LC3I to LC3II in HEK293T cells where you see the double band. The fact that your antibody can detect two bands does not mean that it works properly. I have tried different antibodies for autophagy and the only one that works in excellent way is the anti-LC3B #2775; Cell Signaling Technology. You can see in my recent paper how clear is the signal and the conversion between the two forms. The paper is Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes. (Figure 2A). Good luck, Giulio
Hello, As first thing I would verify how good is your antibody by treating with Rapamycin or others autophagy inducers to check if you can detect the conversion from LC3I to LC3II in HEK293T cells where you see the double band. The fact that your antibody can detect two bands does not mean that it works properly. I have tried different antibodies for autophagy and the only one that works in excellent way is the anti-LC3B #2775; Cell Signaling Technology. You can see in my recent paper how clear is the signal and the conversion between the two forms. The paper is Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes. (Figure 2A). Good luck, Giulio
Hi JIngting,
I can recommend you this antibody. Send you the link:
http://www.cellsignal.com/products/primary-antibodies/lc3a-b-d3u4c-xp-rabbit-mab/12741
Regards,
Monica.
Hi JIngting,
I can recommend you this antibody. Send you the link:
http://www.cellsignal.com/products/primary-antibodies/lc3a-b-d3u4c-xp-rabbit-mab/12741
Regards,
Monica.
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Hi Giulio,
Very thanks to your reply! I've treated HEK293T cells with 100nM rapamycin for 1 or 5 hours and I can see the obvious conversion of LC3I to LC3II. So I think it can work properly. The antibody I used is from MBL. I just wondering why I can detect it in HEK293T cells but not in Jurkat and Raji cells.
Hi Giulio,
Very thanks to your reply! I've treated HEK293T cells with 100nM rapamycin for 1 or 5 hours and I can see the obvious conversion of LC3I to LC3II. So I think it can work properly. The antibody I used is from MBL. I just wondering why I can detect it in HEK293T cells but not in Jurkat and Raji cells.
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VOTE
Hi Jingting
For a start, it seems you are not the first to have this problem (see link).
Although I have never used these cell lines, I would recommend blocking the late stages of autophagy using bafilomycin A1 (10 nM for 2 hours for example) or chloroquine. If the turnover of LC3 is very rapid in these cells, you might have to block autophagy to accumulate detectable levels of LC3. You can also combine this treatment with an inducer such as rapamycin (personally I use Torin1 at 250 nM for 3 h with good effect) to further increase LC3-II levels.
https://www.researchgate.net/post/Difficulty_detecting_LC3_in_Jurkat_cell_lysate
Hi Jingting
For a start, it seems you are not the first to have this problem (see link).
Although I have never used these cell lines, I would recommend blocking the late stages of autophagy using bafilomycin A1 (10 nM for 2 hours for example) or chloroquine. If the turnover of LC3 is very rapid in these cells, you might have to block autophagy to accumulate detectable levels of LC3. You can also combine this treatment with an inducer such as rapamycin (personally I use Torin1 at 250 nM for 3 h with good effect) to further increase LC3-II levels.
https://www.researchgate.net/post/Difficulty_detecting_LC3_in_Jurkat_cell_lysate
More
VOTE
Hello,
As first thing I would verify how good is your antibody by treating with Rapamycin or others autophagy inducers to check if you can detect the conversion from LC3I to LC3II in HEK293T cells where you see the double band. The fact that your antibody can detect two bands does not mean that it works properly. I have tried different antibodies for autophagy and the only one that works in excellent way is the anti-LC3B #2775; Cell Signaling Technology. You can see in my recent paper how clear is the signal and the conversion between the two forms. The paper is
Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes. (Figure 2A).
Good luck,
Giulio
Hello,
As first thing I would verify how good is your antibody by treating with Rapamycin or others autophagy inducers to check if you can detect the conversion from LC3I to LC3II in HEK293T cells where you see the double band. The fact that your antibody can detect two bands does not mean that it works properly. I have tried different antibodies for autophagy and the only one that works in excellent way is the anti-LC3B #2775; Cell Signaling Technology. You can see in my recent paper how clear is the signal and the conversion between the two forms. The paper is
Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes. (Figure 2A).
Good luck,
Giulio
More
VOTE