I have been trying to overexpress c-Myc and MAX in HEK 293 cell either alone or by co-transfection. c-Myc is over-expressed with high signal. However MAX has very low signal. I tried to check the constitutive expression in breast cancer and colon cancer cell lines as well. c-myc is detected in some of the cell lines with very good signal. However I am not being able to achieve a clear band of MAX in Western blot. I use Goat anti Max ANTIBODY for Max , and also flag tag antibody while over-expressing in pdream vector, in HEK 293 cells.
I would be grateful for any suggestion. Please help me out. I forgot to mention I am working with human c-Myc and MAX.
Some random suggestions:
1. If the FLAG tagged version isn't detectable in 293 cells, I would be concerned that there is an error in your plasmid. If you haven't already, make sure the sequence of the gene is correct, that it was subcloned correctly, that there is a good Kozak sequence, etc.
2. Poor constitutive expression might indicate that Max is simply a low abundance protein, but the antibody you are using might also be poor. Try an antibody from a different source.
3. For the western, try the most sensitive developing reagent you can get. With reagents such as ECL ultra, a lot of things might show up that you don't see otherwise.
4. The protein might be expressed, but not extracted effectively from the cells with the buffer you are using. This can especially be an issue for nuclear proteins. One approach is to lyse the cells in 1X loading buffer (with 2% SDS and a reducing agent). Then pass the lysate through a 27g needle to reduce the viscosity and boil. Alternatively, try RIPA buffer with 0.5% DOC and 0.1% SDS.
5. You could try to improve the efficiency of transfection with different reagents, more DNA, etc. I recommend overnight transfection of 293 cells in media with 10% FCS, but no Pen Strep, using Fugene or a similar reagent.
Good luck!
Thank you Michal Jeremy for your response. Considering first suggestion, I have confirmed my DNA sequence and its fine.I have used two types of antibodies flag tag and also max specific. The flag tag is fine with other protein so I think that may not be a problem. May be I have to use other more sensitive reagents for detection as you have suggested in point no three.
I use lysis buffer Tx-100. I have been using 2%SDS and beta mercaptoethanol before I boil, then I load the sample. As you have mentioned I shall try to pas through the needles which I have never done.
Furthermore, I use Fugen 6 for transfection, but I have been using media with Penicillin plus streptomycin 1%. So I will rectify when I try next time.
Thank you for your suggestion and time.
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