Hi
I am trying to get protein expression of a protein called AVPI1. The size of this protein is quiet small-17kda.I have tried western blot for this protein twice- once with 20mcg of protein and second time with 40mcg of protein but still does not get any expression. So there could be few possibilities of why this is happening:
i) The antibody is not good
ii) The cells express AVPI1 at mRNA level but at protein level the expression is minimal that it cannot be detected
iii) This protein is too small to transfer at 20V.
I think I need to optimize the conditions before concluding that the antibody provided is not good. Here are the experimental conditions I have used:
i)
I used 4-12% pre made gradient gel https://www.thermofisher.com/order/catalog/product/NW04120BOX.
ii)The gel was transfered using iBLOT gel transfer stacks(https://www.thermofisher.com/order/catalog/product/IB301032) in iBLOT gel transfer device. The transfer was carried out at 20V for 10 minutes.
iii)I am using nitrocellulose membrane of 0.2 um pore size. Do you think I should use smaller size than this?
iv)I used 5% skim non-fat dry milk in 1x PBS for blocking.The blocking was done for an hour at RT.
v) I used anti-rabbit and anti-mouse secondary antibodies raised in goat. (IRDye® 800CW Goat anti-Rabbit IgG (H + L), 0.5 mg and
IRDye® 680RD Goat anti-Mouse IgG (H + L), 0.5 mg)
The blot was scanned using Odyssey scanner. The results showed bands for control antibody- beta actin, but no bands for my target protein.
Any suggestions on changing any conditions?Thanks for your time.
Hi Jyoti,
I have previously worked with a protein which was very lowly expressed (often not expressed at all) and approximately 9 kDa in size and it took a lot of optimization! I used the following conditions for my western blots:
- Load between 20-30ug of protein
- 12 or 14% gel. and run the gel only 85% of the way
- Transfer to PVDF membrane using the following conditions: 300mA for 45-60mins (wet transfer system).
- I then manually developed the blots and often did longer exposures of up to 10minutes.
- I also did not probe for the beta-actin and my target at the same time as the signal from actin was too intense at such long exposures and my target bands were not detectable.
I ensured my positive control (extracts from rat hypothalamus which highly expresses this protein) was also included at all times as it was the only way to ensure there was no antibody issue.
I hope this is helpful!
Hi Jyoti,
To add my experiences with a small protein (10kDa):
- 17kDa is not too small. So I would expect there is an issue with expression/ blotting/ detection.
- I blotted successfully using a PVDF membrane.
- I used a Schaegger gel (basically tricine instead of glycine) at 16%. It usually yields better results for small molecular sizes.
- as Kate pointed out, results were better if the gel was not run for too long. I used a visible ladder and let the 10 kDa marker band run to about 50%
- to reliably detect my protein, I removed the upper part (high molecular size) of my gel, as it carried a lot of unspecific signal (my antibody was not too good either).
- An antibody service provider told me that peptide-based antibodies (the making of which requires no additional work from you) work better for small proteins.
Hi Jyoti,
I would also recommend, as Kate mentioned, to use a self-made gel with a higher percentage. In my last onset we had to difference between a protein with two bands, one at 16 and one at 18.
Following onset was sucessfully used:
- We used a 15% gel (12% should also work in your case)
- 30-50ug protein
- high voltage (120) at the beginning for the collection gel, as soon as the samples are in the seperating gel decreased voltage to 50-80V
- the gel will run about 3h
- also stop after 3/4 are reached
- wettransfer over night at 80V on PVDF membrane
- using ECL-Plus to increase the signal
- developing between 5-10min
Good luck!
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