Hi,
I am trying to blot a protein of 8kDa with western blotting. So far I have tried to use 0.45um & 0.2um nitrocellulose membranes for transfer as well as 0.2um PVDF.
I have tried to use different voltage & time when running the gel, always with 4-20% pre-casted gradient gel from Bio-Rad, as well as tried different transfer parameters, voltage, time & buffers but only semi-dry transferring though.
I have ponceau stained after transfer and I can see that there is no transfer of proteins below 20 kDa.
I really need to make this work and would be very grateful for any input.
Best,
Linn
In case this could help you, for SDS-PAGE I use 16% acrylamide gel and I usually perform the proteins transference by diffusion to two nitrocellulose sheets (placed on both sides of the gel), for 18 hours. The nitrocellulose membrane that I use is Supported Nitrocellulose Membrane, 0.45 µm (Bio-Rad).
You could also try with Tris-Tricine SDS-PAGE [Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987;166:368-79].
Dear Linn, as Maria Luisa pointed above Tris-Tricine gels seems to be answer. Maybe you could find extra tips in the following links (first one is an older RG thread about this same topic):
https://www.researchgate.net/post/How_do_I_run_very_low_molecular_weight_proteins_on_an_SDS_PAGE_gel
https://www.ptglab.com/news/blog/tech-tips-in-search-of-low-molecular-weight-proteins/
https://bio-protocol.org/bio101/e3093
Good Luck
Hi, can I ask if you check your gel with coomassie? The low molecular proteins were on the gel? Or not?
I would also propose to use a higher concentrated (maybe a gradient~) SDS gel as mentioned above (e.g. 16%).
I use bis-tris gels, 4-12%, transferred to PVDF using an iBlot2 for 6' at 20V ( I think it's 20V). The problem with small proteins is they usually transfer out quickly and then go *through* the transfer membrane. I've never had a small protein not transfer out of a gel if the larger proteins transferred. Did you ponceau your blot or your gel?
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