Dear all I am desperately trying to show an increase of h3K4 2me in my protein samples after incubation with an LSD1 inhibitor. I am working with AML cell and my results are variable all the time. Any suggestion/tips troubleshooting.
To my mind, you should first design a good positive and negative control to be sure that your method works correctly.
As we already discuss in a precedant post (I remember it) histone purification will help you a lot.
Can you tell us more about your assay design ? And where did you buy the assay incredients ?
After having the same problem for months, a colleague suggested using 1x SDS-loading buffer to harvest the cells followed by a sonication step. So instead of lysis buffer, I simply add SDS Loading buffer to the cells (in my case macrophages), pipette up and down (the lysate becomes very viscous). Then I sonicate for 10 seconds (5 sec on, 5 sec off) at level 3. This is enough to release the histones from the DNA which will make the Western blot comparison a lot more consistent between samples of each biological replicate, as well as different replicates. I did this to check H3K4me2, H3K9me2, H3K9me1, H3K27me2, H3K27me3, H3K4me3, H3K36me2 to compare WT macrophages with different knock-downs and my results have been consistent.
Note that I tried various histone extraction methods before this, and they didn't work for just doing western blots because the level of extraction wasn't the same for each sample at different biological replicates.
Hope this helps.
I agree with Dr. Ozgun Erdogan about harvest the cells directly with 1x SDS-loading buffer. In that case scrapping on ice and heating quickly the samples to denature proteins and minimize protease activity.
Hope this helps too.
Thank you Dr Ozgun Erdogan `and Victor. Do you use any Protein inhibitors? I think I will give it a go! How many cells do you use per ml? Thank you very much!
Yes, I use a cocktail of protease inhibitors (calbiochem 539134) but you can obviate it if you scrape quickly the cells with 1x SDS-loading buffer on ice and immediately boil the samples. The amount of cells depends on your protein abundance and I have not experience with H3K4. Particularly, I use 8e10+6 cells per mL the most of times.
When using SDS-loading buffer I don't use protease inhibitors if I do everything quickly. SDS denatures the proteins in the lysate so you really don't need inhibitors if you sonicate and/or boil right away.
I tend to use 200ul/well for 12well plate, 500ul/well for 6 well plate and about 1ml/plate for 10cm plate for macrophages (you probably would want to use a little less volume for HEK293 cells, less number of HEK293 cells exist per surface area. So maybe 150, 300, 600 ul respectively might be a good start). Then I use 10ul per sample (or lane) for loading on the SDS-PAGE gel. Histones are highly abundant and this amount is enough for histone western blots. However if you would like to blot for other cellular proteins to check a specific phenotype you might want to double the amount you load on the gel.
Good luck!
Note: I would definitely recommend the sonication step right after harvesting for consistent results between different biological replicates.
Hi Ozgun Erdogan. I am trying to use your suggestion for the next Blot. Last question: For the SDS loading buffer I have the 3x loading buffer. You use this one diluted in PBS or you use an home made one? Thank you very much.
I just dilute my 4x loading buffer stock (+BME) 1:3 in water to make it 1x. Good luck!
Ozgun Erdogan, Thanks for the help....Can I ask you as well how long you transfer for and at which voltage? We have very similar cells (Using AML cell line, THP-1)..Thank you!
Well, I run my samples in 15% SDS-PAGE gel. Since it is 15%, I run the gel 80V for 20 mins for stacking, then 150V for 60-70 mins. I also check frequently to make sure I don't run it for too long (check 15KDa band on the marker).
As for the transfer, I transfer in 300mA (constant amper)(We use PVDF membranes, more sensitive than nitrocellulose) for 100-120 mins (depending on how fresh the transfer buffer is, if it is fresh buffer 100 mins should be enough) on a stir-plate in the cold-room to keep the system as cool as possible. I use milk to block for 30-45 mins at Room Temp.
If you are using the Abcam antibody (I don't have the lot#), note that H3K4me2 or H3K4me3 antibodies are very very strong. So I would try primary antibody incubation for 1 hr, and keep that batch of primary antibody in a 15ml tube in the cold room after that incubation (you can re-use it to try overnight rotating at cold room if the signal turns out to be weak). Wash 3x5 mins with TBST, then do secondary antibody incubation overnight at cold room.
I tend to do either primary or the secondary antibody incubation overnight at cold room, and the other incubation at room temp for 1 hr. i.e. If the primary antibody is too strong, I do primary for 1hr at RT & secondary overnight at cold room; if the primary antibody is not very strong, I do primary incubation overnight at cold room & secondary for 1 hr at RT. So you are welcome to try both ways.
Does this help?
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