Hi all,
I have problem with Stathmin1 17 kDa protein from mice brain tissue on western blot. Does anybody have experience with Western blott for 17 kDa protein? I am using 12% gel, SDS-PAGE, and then transfer to PVDF membrane. In gel electrophoresis we use 60 V for 30 min and after that 120 V for 1 hr.
Wet transfer is 120 V/2hrs/ice. I have attached the best photo of my experiment. Primary antibody: Rabbit polyclonal, Abcam.
Could you please mention the marker size of the image you posted here? What is the protein that appears on the membrane?
Maryam Karamikheirabad
Hi Mostafa, thanks for asking. The marker is 15 kDa. the protein that you are seeing above of it is Stathmin1.
Hi, Maryam,
Excuse me, I would like to ask you what is exactly your problem? Is the image correspond with SDS-PAGE or with the blot? Are the clearer bands the 15 kDa marker?
Hi Maria, Thanks for your consideration. This image is taken after blotting the membrane with "Fusion" software. Yep, the first is marker, then 3 protein sample , then again marker and next 3 protein sample, lastly again marker. It mean I have duplicated my samples. My supervisor likes sharper bands and also not curved like that in first lane.
Bands look fine and appropriate, anyway, You may have a look at the links below for improvement. These are good sources to follow;
- https://www.bosterbio.com/western-blotting-troubleshooting-guide-download-page
- https://www.bio-rad.com/en-uk/applications-technologies/western-blot-doctor-protein-band-appearance-problems?ID=MIW4Q8C4S
Maryam Karamikheirabad
I am still not sure what the problem is that you are facing. Since the bands are respective to your protein as you mentioned, could you please specify the problem again.
Mohd Najib Mostafa the first lane is marker, then 3 protein sample , then again marker and next 3 protein sample, lastly again marker. It mean I have duplicated my samples. My supervisor likes sharper bands and also not curved like that in first lane.
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