I am using 80V/90min to transferring LC3 from 12% SDS-PAGE to PVDF membrane. But recently, I have not gotten any band of LC3. As I've read somewhere that LC3 could swim through the membrane if we use long transferring time and high voltage.
Could you please suggest me the appropriate voltage and time for transferring with 1 and 2 membranes?
Hi,
3 mA per cm2 of PVDF membrane and 25V for 1h.
I am using also transfer buffer with 10% methanol.
It works.
I set I not U but I hope it will help.
Thank you, Edyta.
I also will try with a new antibody to ensure that it works.
what you use for transferring the gel? transbolt system or wet tranfer or semidry? for semidry and transblot system u can use 250 volt for 1h30min... and for wet tranfer system you can use 150 volt for 1h 15min..... You will get good result but please do dye out because LC3 is low kDa protein
We are using wet transfer, 80V for 1h30 using 10% methanol on nitrocellulose membrane not supported from BioRad. It works perfectly for LC3 immunoblotting. Best is to look for the exact membrane reference you are using what the company recommend.
Using 2 membrane can be used to control that the proteins do not traverse the first membrane, but it gives bad results if you detect the extra membrane.
What about a positive control like cell treated with rapamycin (cf file attached). if no cell culture available you may buy cell lysate
http://www.abcam.com/autophagy-control-hela-chloroquine-treated-and-control-lysate-ab171461.html
You can also check if the transfer worked correctly using ponceau or amido black staining.
If you see bands under 20kDa you can check the quality for example by trying to detect monomer of ubiquitin
LC3 I and II are below 20 kda and usually will run off of a 12% gel. I would suggest you use a 15% acrylamide gel to separate LC3. See the attachment I have included from one of our publications.
The western blot in the image was transferred overnight at constant voltage (30V).
Hi Michelle do you use nitrocellulose or pvdf and is this a wet transfer?
Hello Yudhishtar,
I typically use nitrocellulose (0.2 um) and I always use a wet transfer.
Awesome suggestions on this query and I totally agree with the points mentioned by Dr Casadei. I would like to add the following couple of points:
1. Running the samples on relatively low % gels such as 12% or lower can result in diffusion or dispersal of the low molecular weight proteins - which will lead to no bands in WB, For LC3 separation, 16% gel is an ideal choice and once the gel is run, it should be rinsed properly to remove the entire running buffer/SDS from the gel before moving to transfer step.
2. Voltage for the transfer should be kept low to prevent the LC3 I and LC3 II from crossing the membrane. Also, after performing the transfer, researchers should use Amido black or Ponceau S staining to visualize protein transfer and to ensure that the proteins have actually transferred in the low molecular weight region (15-20kDa range).
3. A positive control lysate of serum starved or chloroquinine treated/untreated cells showing extensive vacuolization may be used to check the integrity of test samples. Novus offers ready to use HeLa Chloroquine Treated / Untreated Cell Lysate (NBP2-49689) and Neuro2a Chloroquine Treated / Untreated Cell Lysate (NBP2-49688) for use as controls in LC3 analysis.
Troubleshooting Tips and Technical Answers to more than 25 similar frequently asked questions/ FAQs on LC3 /Autophagy analysis may be found at: http://www.novusbio.com/support/faqs-autophagy-and-lc3
20% Methanol-containing transfer buffer
125mA/ PVDF membrane
for 80 min
Hi,
I'm so lucky to just read your two wonderful papers published on CEA about autophagy.I'm doing the western blotting for several times of LC3 on A549.However, no band of LC3 is showed.So would you please tell me your western blotting conditions including running condition and transferring condition for LC3? Waiting for your reply! Thank you so much!
Hello Xin Zhang,
Thanks for your question.
I loaded >30 ug protein for 1 well on the 12% SDS-PAGE gel.
For transferring, I used Constant 80 Volt in 90 min, PVDF membrane activated by Methanol for 5 min. The transfer buffer contained 20% Methanol without SDS.
I detected the bands using gel-doc system. You may consider to use a good ECL solution for detection. Previously, I recognized that the ECL solution was the problem.
Good luck!
Duy
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