Rat heart tissue, I am assaying for CSE/CTH protein expression always give a triplelt bands at 72,56 and 45Kda (Published data only showing a single band detected at 45Kda in heart tissue).
The antibody, I am using (CSE/CTH :GeneTex: GTX113409) can only detect a band at 45Kda.
The conditions I used for WB as follows: 10% gel, 40V for 1 hour staking gel, 120V for 2 hour in separating gel, primary antibody overnight (1:1000/1:3000 in 5% milk+TBST),Secondary (1:1000/ 1:3000 in 5% milk+TBST) for 1 hr.15min X4 TBST washing in-between primary and secondary antibody incubation.
Attached is the image for x-ray film. Your suggestions will be highly appreciated.
Dear Charith Wijerathne, I have a few suspected cause and possible ways you could try to optimize or troubleshoot.
1. The most likely cause is a post-translational modification (PTM). You could find out from the literature if your protein of interest undergoes any PTM and what conditions could lead to that. Perhaps, your experimental design promotes PTM in your protein.
2. It could also be due to proteolysis. You might want to review how you stored and handled your sample before the normal temperature-degradation step.
3. It could be that you loaded too much protein sample. What you could do is to run a decreasing amount of protein sample and see if the triple bands will appear across the lanes regardless of the amount of protein loaded.
4. Can your protein exist in a complex with another protein? If that is the case, I suggest you boil your protein sample for up to 10 minutes at 95 degrees Celsius in lysis buffer which contains beta-mercaptoethanol (if that is not your protocol).
5. Your primary antibody might be too high and could bind non-specifically to other unblocked proteins on the membrane. I would suggest you reduce the primary antibody concentration.
If it still persists, you could run the same sample, block and then incubate in secondary antibody without incubating in the primary antibody. If any band appears, then it is not the primary antibody but probably the secondary antibody.
From your description. it seems you did not wash the membrane after the secondary antibody treatment. If not, do not forget to wash 3x after the secondary antibody treatment as well.
What do you mean by 1:1000/1:3000?
If you just reduce the amount of loading protein, you may not see your protein of interest as the 45KD is a lot thinner than your non-specific protein bands. So, troubleshoot the antibody dilution and washings after 2nd ab incubation.
Chandra Somasundaram .Thanks for your suggestions.
The membranes were washed 15minX4 after secondary antibody incubation as well. I have used different 2nd antibody dilutions (1:1000 and 1:3000).
- Badges
- Science topic