We are trying to run a blot for LC3 [MAP1LC3B], however unlike the two 14 & 16 KD band we are getting only one band (picture attached). This blot has 5 treatments: Lane1: Control, Lane2-5: Treatment in increasing order Cell used for this expt: HEK293 Gel % = 10% in 1mm spacer glass Development: Manually in Dark Room on X-ray film Primary Antibody dilution= 1: 1000 (as recommended), overnight treatment at 4C Secondary Antibody dilution= 1;10,000 (recommended= 1;10,000 to 20,000) 1 hr at RT Chemi-luminescent: ECL plus 1:1 in 1 ml H2O Please kindly assist why we are getting only one band in all the samples. Also why all the samples seems to be attached with each an other and why they appear zig-zag is structure rather than straight
10% gel could be the issue. The band probably ran out. Try 15% gel or gradient gel (4-20%).
You need to have gradient mixer. Alternatively, its available in Bio-rad as precast gel.
If u have a protocol for prepration of gradinent gel can you send me
You can try this video. https://www.youtube.com/watch?v=zu5a-kpMK8k
PS: I never make it by myself.
Hello :)
I also use LC3:)
1. Try 15 % Ge
2. Try to use 120 V for 4 h (start with 85V for the first hour)
Then you should see both.
#Zi Wei thank you for this information it is very helpful for me
Amit Kumar Shrivastava Use 12% gel and run your gel at 70V. Do not run the gel its entire length. Stop running when the dye front is around 1.5 cm above from the bottom of the gel. The LC3 bands will not become fuzzy in this way.
Use 0.2 µ PVDF membrane for transfer instead of 0.45µ Nitrocellulose membrane.
I'm writing this here in case it helps someone. I struggled with one band for a while. I made the following changes, and then I got 2 bands:
1) 4-20% gel
2) Changed nitrocellulose to pvdf
3) Reduced the voltage to 90
4) I did a quick transfer as I was worried that I was losing the LC3-II band due to over-transfer.
I hope it helps
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