Hi all,
I have been attempting to blot for LC3I and II and i have been having a strange issue, the bands seem to be showing up around 23 kDa. I have done the blot twice now and still the same issue, except on the second i included a 4-hour starved control. Due to the pattern of staining for my starved control, the bands seem correct. I am wondering if my lysis protocol is leaving them attached to some larger structure?
My summed up lysis protocol: 30min in NP-40 on ice with vortex every 10min, followed by 20min spin at 14000 RPM, 4 degrees
Blotting protocol: 20ug protein per well, 80V for 30 min then 185V for 40 min. Low MW turbo transfer 5 min, 1 hour blocking using BSA, overnight primary @ 4 degree 1:1000 (ABCEPTA rabbit), 6 washes (3 for 10min, 3 for 5 min), 1 hr secondary (1:2000), same washing, Pico chemi and image
Blot map: Starved control is in last lane. Uninfected cells in first lane (J774s)
ladder is precision plus dual color, my LC3 bands appear to be showing up between 20 and 25 kDa
Hi, Caleb!
Have you checked the concentration of your gel and acrylamide you are using? Maybe gel is too concentrated. What is the percentage of your gel?
Angélica Lauria i used a 4-12% NuPage bis-tris gel, sorry not sure why the tag didn't work before.
Most LC3 antibodies detect that non-specific 20ish kDa band. The only way to get rid of it, is by cutting your membrane. That is not LC3. Possibly you have low LC3 expression/transference and that's why you only detect the non-specific band. See the images here: https://www.mblbio.com/bio/g/product/autophagy/pickup/lc3.html
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