Hey guys, I am having an issue where I am not getting the correct results on my Westerns that I am running. When I run my Western for VGLUT1 from mouse hippocampi samples, with an antibody catalog #48-2400 from Thermofischer, I consistently get bands in the ~300kDa area. I am trying to troubleshoot the issue and I got a new lot # antibody from Thermofischer but the results are the same, I still see a 300 kDa band. You can see in the 9/28 Western Result 1 that I have attached in the post. After running it with VGLUT1, I ran it against b-actin to see if there was any protein present incase I might have made a mistake with running my gel but as you can see in my other image, the B-actin shows perfectly fine even though my image is overexposed. My basic protocol for Westerns is:
1. add 1x loading buffer (SDS) to my sample.
2. denature proteins @ 90 degrees for 5minutes
3. load sample and run in 12% SDS page gel.
4. Transfer onto a nitrocellulose membrane.
5. block with 2% Milk in TBST for 1hr @ RT
6. Add primary Rabbit anti-VGLUT1 antibody @ 1:2500 for 12hrs in 4 degrees
7. Wash
8. Add secondary antibody anti-rabbit IGG conjugated w/ HRP @ 1:2000 for 1hr @ RT
9. Wash
10. Add detection reagent and image
Based on the product, I should have a band show up @ 61kDa but I am seeing it at 300kDa. I was considering running another Western with the same samples but not denaturing the proteins to see if there is any difference in result but I was hoping for some other suggestions incase I might be missing some key component. Thanks guys
From your keywords I notice that you use nitrocellulose membranes. These are only second best, PVDF has higher protein binding affinity and capacity and is mechanically stronger.
However, if a 62 kDa protein appears at 300 kDa (i.e., does not enter the gel), then it is aggregated. The problem hence is with your sample preparation. As you say nothing on how you prepared the brain samples, I cannot comment. Some hints on electrophoresis though:
In general, especially with transmembrane proteins, heating to 60 °C for 10' is better than boiling. I would also check the sample buffer, maybe you made a mistake there. How do the gels look (use Zn/imidazole staining, which is cheap and doesn't interfere with western, doi:10.1016/j.ab.2004.02.017)? Do you get good bands throughout the lane?
Once electrophoresis works, check the blotting procedure. Do the bands transfer out of the gel? Can you detect the transferred bands on the membrane (Ponceau staining)?
One last, more fundamental point: In science, you should not "desire" a result. An experiment -- if correctly performed, which is unlikely here -- is a question to nature. Nature will answer in its own way. "The Logic of Scientific Discovery" by Karl Popper (ISBN 978-0-4152-7844-7 ) is the key text here.
Engelbert Buxbaum
Firstly, there is no mistake in any buffer since I re-ran it with b-actin and the bands showed properly which in my mind means that every solution I made was sufficient enough to work. Secondly, I did get good bands throughout the lane after gel electrophoresis and as stated in my original post, the bands transferred out of the gel correctly since I did run it in b-actin and bands with the correct band size, namely 41 kDa, showed itself.
Hi Jasper, endogenous lipids in the brain sample could be interfering with the SDS-PAGE of integral membrane proteins. There might not be enough SDS in your sample buffer to compete out the endogenous lipids.
Actin is not lipid associated so it wouldn't be affected.
If this is going on, you could do serial dilutions of your sample into SDS sample buffer or use 10X SDS sample buffer and see if your protein runs differently with increased SDS.
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