Do you have any suggestion how to get rid these non specific bands?
These are my protocols:
1. Osteoblast cells were cultured in 100-mm cell culture dishes in serum-deprived α-MEM overnight.
2. TNF-α were then added to the dishes for specific periods (0, 6h, 12h, 18h, and 24 h) in serum-deprived α-MEM.
3. Then, washed twice with ice cold PBS and lysed using RIPA buffer (Millipore, Burlington) containing 1% protease and phosphatase inhibitor (Thermo Fisher Scientific, Rockford,).
4. Protein were treated with β-mercaptoethanol and laemmli sample buffer (Bio-Rad, CA) and denatured at 95 ◦C for 5 min as a preparation for SDS-PAGE.
5. 50 ug were loaded into gels 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad, Hercules,) and transferred to a PVDF Trans-Blot Turbo Transfer System (Bio-Rad, Hercules).
6. The membranes were blocked in Block-Ace (DS Pharma Biomedical, Osaka, Japan) for 1 h at room temperature and were probed AGTR1 Rabbit polyclonal Ab, phospho-SAPK/JNK (Proteintech; 1:1000 dilution) overnight at 4◦C.
7. The membranes were washed in TBS-T and TBS, then incubated with anti-rabbit IgG HRP-linked Antibody (Cell Signaling Technology, MA, USA; 1:5000 dilution) for 1 h at room temperature.
8. The membranes were washed in TBS-T and TBS again, then incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL, USA).
Here I attach the figure. The protein that I want to get has MW of 41 kDA.
Thank you so much for your kind help.
Hello
There are many reasons for the presence of non-specific bands on the membrane.
1. Try to reduce the amount of protein loading from 50ug to 30ug unless the target protein is present in low abundance. This is because excess amount of lysate loaded will cause antibody to bind non-specifically to proteins which is in excess resulting in multiple bands.
2. You need to increase the dilution of the primary antibody. You can go upto 1:4000. Also, the secondary antibody concentration is too high. Try secondary antibody dilution of 1:15000 to 1:20000. Using high concentration of antibodies either primary or secondary can lead to formation of non-specific bands.
3. Incomplete blocking can cause primary and secondary antibodies to bind to the membrane causing non-specific bands. Try to increase the blocking time from 60 mins to 90 mins.
4. Use of polyclonal antibody could also cause multiple bands because of its ability to recognize multiple epitopes. Try using monoclonal antibody, but keep that as the last option.
5. Make sure that the antibodies are not contaminated.
6. Poor handling of the sample which could lead to sample degradation should also be considered.
I hope this is helpful.
Best Wishes.
Thank you again for your suggestion Prof Malcolm Nobre . I will try to reduce the amount of protein to 30 ug, increase the secondary antibody dilution, and increase the blocking time.
Overnight blocking with 3-5% BSA/skimmed milk with 0.2% v/v Tween 20 at ~4C greatly improves antibody selectivity.
Hello, I need some advices again.
I already repeated twice, and these are the results.
I attached 3 figures from three membranes. I already add number in that figure.
1. First picture is my original membrane, the same figure like before
2. Second picture is my second trial.
- Lower protein concentration (30 ug)
- 1st ab dilution to 1:1000
- Reduce the 2nd ab dilution to 1:5.000
- Longer blocking time 4 degree celcius overnight.
It is getting better but I lost the differences of the band intensity compare to my first trial, the first column should be different with the last column (red boxes). But it all looks the same.
Do you have any suggestion why it gave me different results?
3. Third picture is my third trial.
- Lower protein concentration (30 ug)
- Reduce 1st ab dilution to 1:2000
- Reduce the 2nd ab dilution to 1:5.000
- Longer blocking time 4 degree celcius overnight.
I still found the non specific band. Any suggestion? and I also lost the differences of the band intensity (red boxes).
Thank you
these are way too many bands to be called non-specific. What seems the most obvious to me is that your blocking did not work properly. Increase the blocking, and maybe do not wash your blocking too harshly. Use PBS pH 7.0-7.4 to make blocking solution, do not wash more than twice, and keep it on a rocker to wash and don't manually shake it to remove excess blocking. The fact that only bands and not the spaces between them are lighting up means that the antibodies are fine.
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering
- Membranes