Hello all
I tried to transect MCF-7 by lipo 3000 using 250 ng, 500ng or 2000ng plasmid DNA for 48 hrs in serum-free media. But unfortunately not able to successfully transfect my plasmid. Secondly, I saw no. Of non-specific bands on my blot from. Here I have several doubts.. 1 is my transfection technique Has issue? 2 or optimization is needed for western blot if yes then what could be the conditions? 3 my primary antibody has issues or it is degraded ( sc-7392)?
Lysis was done in 1%NP-40 buffer, 150 mM NaCl, 1 mM EDTA ( 30 min on ice with 30sec vortexing in every 10 min) centrifugation 14000 rpm 4C for 20 min
Loaded: 30 ug
Blocking : 2 hrs RT 10% BSA in 1x TBST
1 ab: O/N 1:3000 4 C in 1x pbst
2 ab: 2 hrs secondary mouse (1:5000)
Hello Suchi
I am concerned that the dilution ratio of the antibody is lower than the recommended concentration.
However, since the detection system using HA-tag in Western blotting is highly sensitive, we believe that this level of detection is possible.
https://datasheets.scbt.com/sc-7392.pdf
HA-probe (F-7) is recommended for detection of proteins containing the HA tag by Western Blotting (starting dilution 1:200, dilution range 1:100-1:1000),
Best
Hello Sir, I am agree with your concern.. But the problem is as soon as I am increasing the dilution no. Of non- specific bands are increasing this is making more worried..
I not able to understand where is the problem transfection or western blot.
I don't have any other considerations other than this. If you doubt the efficiency of transfection, you should try transfecting GFP-expressing vectors.
I also wonder about the plasmid construction process.
As you may already know, the following paper used this antibody to detect proteins in MCF-7.
https://cancerres.aacrjournals.org/content/early/2012/06/18/0008-5472.CAN-11-3832
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