For this western blot, i am running the tricine-sds-page. I run the gel until the 10KD band of the ladder has traveled half of the gel. But, when i go for the commassie or silver staining, i dont see any band after the 10KD band. Lower molecular weight proteins does not appear. Should, i proceed for the western blotting ? For this tricine-sds-page, i am following the nature protocol (Schagger 2006)
Dear Ibrar, are you using transgenic mice model that over expresses human APP? In case of WT mice, I double if you could find any detectable amount of Abeta if you're not using Pico, Femto, or Luminata substrate.
Apart from APP, there are several different aspects one should consider to get the monomeric bands of Abeta and reproduce the results. Here are the following parameters you might want to consider:
- Tricine gels are good, but you don't always need to use them to detect Abeta, I use NuPAGE 4-12% gradient precast gels that works quite fine.
- Load enough amount of brain lysates so that you have detectable amounts of amyloid beta. Otherwise, you shall opt for immunoprecipitation to improve the amounts of Abeta in the samples you load.
- Use 0.45 µ membranes, both Nitrocellulose and PVDF works fine.
- After the transfer, boil the membrane in 1X PBS for 5-10 minutes that helps to reveal the epitope.
- If you're using 4G8 antibodies, there's no surprise that you don't find the monomeric bands. Using 6E10 antibodies helps.
I hope these points help, try these parameters, if you still find difficulties feel free to contact.
Best regards
Karthik
I am not really into harming animals with experiments, it is not my thing. In addition, it has been demonstrated that having pathology in the brain does not equate with a person necessarily developing dementia. It is more likely to be individual immune response to having pathology in the brain, i.e. inflammatory response but complex with perhaps other causal links. (see the well known Nun's study)
Dear Ibrar.
Karthikeyar's advises are very sound but I do wonder about your research question. For one, detecting Abeta peptides even with 6E10 in brain tissue is a tricky business. Which is why some Urea gels preparations are used, depending in how important is to distinguish between different Abeta species. A western blot will answer to you presence or absence of Abeta in your samples but even in a semi-denaturing western blot you will see Abeta oligomers, is that your idea? Oligomeric Abeta assessment? Finally, if differentiating between species, monomers and oligomers is not your aim but evaluating Abeta concentration is, then ELISA would be a better approach for that.
Best regards
Diego
Dear Diego,
Detection of Abeta using the gradient gels for separation are quite possible, all one has to do is to go through the literature for recommended protocols. Urea PAGE (Klafki's protocol), Tricine gels, Native PAGE are other methods that are specifically addressing for the different oligomeric stages. Yet, if one wants to detect the oligomers from the mice brain lysate, I'd rather recommend to use oligomer specific antibodies (for example, A11) for immunoprecipitation and then detect the broken down monomers as a measure for oligomer content. Relying on SDS-PAGE for oligomers present in the animal/human brain is not appropriate. Having worked with mice, non-human primate and human brain lysates, am sure that one could use get the reliable results if one follows those aspects I have mentioned above.
Best regards
Karthik
Dear Karthikeyan
I am empirically familiarized with gradient gels and Wiltfang protocols for Abeta detection, but thanks anyway. Plus, I would not consider A11 as an Abeta oligomer specific antibody, we have had cross reaction with other oligomeric preparations. Perhaps you should check this yourself, for your peace of mind.
Cheers
Diego
Dear Karthikeyan
Thank you for the response. I have the 5X FAD mouse model of the Alzheimer disease, and i am already done with the silver staining and Thioflavin S staining of the brain sections and also with the ELISA. All results are showing the over expression of the Abeta42. I also want to do the western blot, before it, i tried the tricine gel and it ran nicely which shows the good resolved ladder but after staining with comassea or silver staining, no band appear below the 10KD band of the ladder. I just want to ask, where are the cells all smaller proteins including the abeta42, why they don't appear in the gel like other proteins.
Dear Diego,
Commercially available A11 has got some cross reactivity, sure, but when it comes to oligomer/conformation specific antibodies, A11 recognises other misfolded forms like Alpha-synuclein. There are certainly a few very good conformation specific antibodies I have worked with that are very good.
Dear Ibrar,
Detection of monomeric band of Abeta from the brain lysates using commassie or silver staining won't work. You to specifically detect the Abeta using specific antibodies. As the loads protein that are present in high quantity will easy get stained and the monomeric Abeta that are in traces won't be very prominent. Try Western blotting, that's the way you have to detect.
Thioflavin S is used to stain Abeta plaques and in ELISA you have Abeta specific antibodies, so your approach to detect Abeta with silver staining won't help. Hope this helps.
I am using the Santa Cruz anti-amyloid antibody, these detect the many multimers very nicely but not the monomer. But, when i cut the lower part of the blot and developed it separately, it shows the band at the place of the monomer.
Ibrar,
I have never had much luck with coomassie and Abeta on gels. Silver staining has also been tricky as it seems different oligomers pick the silver up differently. I have seen papers where they seem to get it to work just fine, but I'm not sure what they are doing differently. In my experience the western can pick up what the silver and coomassie cannot. Just make sure you are using an N-terminal antibody.
Wei Hong,
Is it possible that the interaction of the antibody with the abeta is pulling the dimers apart in the IP? That would be my guess.
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