Hello everybody,
I have a problem with western blot of my target protein (the size of protein is around 14 KDa).
I observed band of target protein in SDS-PAGE of samples after 1,2,3 and 4 hours of induction with IPTG. And this band is absent in uninduced sample. But, when I did western blot, I observed a smear.
Does anyone have a solution for this problem?
Hers is other information:
1- The gene has cloned into pET151/D-TOPO. After cloning in TOP10 and sequencing, recombinant plasmid was transformed into BL21. Then I performed a time course of expression. Cell pellets were mixed with 100 micro litter 5x sample loading buffer, boiled for 10 min and 6 micro litter of each samples were loaded in gel (10 %).
2- Blocking buffer: TBS-T + 5% milk powder.
3- Anti-V5-HRP Antibody was used in western blot (1:5000).
4- Washing with TBS-T (2 times and 10 min for each time)
5- 1-Step™ TMB-Blotting Substrate Solution was used at the end of western blot.
hi
1- you can uesd 15% SDS-page .
2- put the gel tank on ice tray during running process.
3- Allow the gel to be fully prepared.
4- test 2ME or DDT in sample buffer .
good luck
Hi Sara Heidarpanah ,
As per your uploaded picture , if feel that your running buffer pH is not proper. (~8.3) just check once before starting Run.
Suggestion:-
Maintaining a constant temperature between 10⁰C - 20⁰C during separation is paramount in obtaining even migration of proteins. Otherwise, outer lanes will move slower than center lanes resulting in artifact known as “smiling”. To prevent this negative effect, you can ensure efficient heat transfer by completely filling the outer chamber of the electrophoresis apparatus with SDS running buffer and constantly stirring the buffer with a magnetic stirrer. Alternatively, heat can be reduced by lowering the running current during separation.
Good Luck !!!
I will recommend you to consider some points to troubleshoot this problem:
1. Make the resolving gel get solidified properly, use clean and dry glass plates.
2. Use freshly prepared buffers in the next time with accurate pH.
3. Add a small amount of reducing agent (beta mercaptoethanol or dithiothreitol) additionally to the loading sample. 15-25 degree Celsius is suggested.
4. Run the gel at lower voltage in the starting phase (100-120V), after the dye migrates from stacking gel to resolving gel, increase the voltage (150-180V) for enhanced resolution.
5. Maintenance of temperature is very crucial for proper migration of proteins.
6. Wash the membrane at least four to six times for better result, you can reduce the time gap (five minutes).
Best regards. Sara Heidarpanah
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