I am trying to measure intrinsic apoptotic markers by measuring the level of Bax and BCL-2 expression in H9C2 total cell lysates. Unfortunately, I do not see any band on the blot. My western blot conditions are as below:
1. Cells lysed with RIPA buffer with Protease and Phosphatase inhibitor added to it.
2. Samples boiled with laemmli buffer for 95degrees for 5mins. Cooled on ice, vortexed and spinned down.
3. Samples mixed with laemmli buffer loaded into 4-20% Tris-glycine gel from Biorad. Running buffer is tris glycinge buffer. Running is carried out at 4degree room.
4. After running, gel is covered with cold transfer buffer for 10mins. Transfer buffer is Tris-glycine transfer buffer containing 20% Methanol, no SDS.
5. Proteins are transferred on to a 0.22% Nitrocellulose membrane. Transfer is carred out in a cold room, 100V for 1hour. After transfer, Ponceau S staining is done which shows good transfer of the lower molecular weight proteins as well as the pre-stained molecular weight markers.
6. Then the membrane is washed with TBST containing 0.05% Tween 20 for 5mins.
7. Blocked with 5% Skim milk in TBST for 30mins.
8. Then the membrane is washed for 3x5mins.
9. Bax antibody (Santa cruz) is added in 5% skim milk TBST with 0.05% Tween20. in 1:200 dilutions for overnight.
10. Next day, the membrane is washed for 3X10mins with TBST.
11. Then secondary antibody is added as 1:5000 dilution in 5% skim milk TBST with 0.05% tween20.
12. Then probed for 1hour at room temperature. Then washed 3X10mins with TBST 0.05% tween 20.
13. Then the membrane is developed with Supersignal west Pico ECL kit from Thermofisher Scientific.
I do not see any bands for Bax at all. Same problem is for BCL-2 as well. This particular cell line do express both Bax and BCL-2 protein. The condition for BCL-2 is similar (1:200 Primary antibody from santa cruz, 1:5000 for secondary).
Please provide some suggestions. I really appreciate your help.
Thank you.
Either you have to increase your protein concentration or you need to make your antibody concentrations less dilute. I don't like Santa Cruz antibodies because they are usually very dirty! I would look for others that work better for detection.
Your WB protocol looks good to me, which suggests that you either have a problem with-
1. Cells - Not enough protein loaded? Or conditions not quite right for expression
Bax will only be expressed if your cells are induced to undergo apoptosis, which you will also see by checking the expression of activated Caspase3 or cleaved Caspase9 for example.
Bcl2, however, you should be able to see in non-treated cells.
2. Antibody
Have you checked that you have the correct species of secondary antibody for these primary antibodies? Are they monoclonal or polyclonal? Have these antibodies been published detecting good expression in this cell line under the conditions used?
3. ECL detection step
Anyone else sharing the kit can verify that its working? How about your detection system hardware?
Can you blot for a highly expressed housekeeper such as Beta-actin?
HI Amanda
Probably you have to increase the concentration of these antibodies at least 2 times. We are using these but we have to increase the concentration of the antibody as well as the amount of protein to 150 ug to detect the bands. A nice approach is using this antibody for do Ip and then western, because these antibodies also have unspecific bands. By doing the IP you clean the blot and concentrate your protein. Good luck
Dear Amanda,
I load around 30ug of proteins. I have checked apoptosis through Annexin V FITC-PI assay. The antibodies are monoclonal and I use the right species of secondary antibody. Beta actin is working perfect. I use LAS400 detection system. These antibodies have previously been cited for experiments on the same cell line.
Sounds like, with all the controls you have there, you have bad antibodies. If you have recently purchased, then you can contact Santa Cruz and they will give you a new batch of antibodies. I've found them to be very reasonable in this regard in the past.
If you'd like to persevere, its worth trying increasing protein level and concentration of antibody, although in my experience 15ug protein and 1/200 antibody has worked.
Either you have to increase your protein concentration or you need to make your antibody concentrations less dilute. I don't like Santa Cruz antibodies because they are usually very dirty! I would look for others that work better for detection.
For WB cells should be at least 10,00000 to 10000000.
Try antibody from Chemicon or Peierce.
You may change blocker from skimmed milk to BSA 3%.
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