I am working with staphylococcus aureus in the lab. I have cloned a target gene with a HA epitope tag and analysed the secretion of this target protein in the supernatant by western blotting using an anti-HA antibody. I also western blotted empty vectors, which didn’t have my target gene and so no HA epitope tag. But when I western blotted the samples, I seen bands around the 47kDa, which I thought were due to my target protein but they were also present in the empty vectors. After doing some research I found that there is a Surface Protein A, which must be binding to the anti-HA antibody. How do I stop these bands from appearing and being able to differentiate my target protein from Protein A? Both proteins are off similar size.
Hi there,
So obviously protein A is present in culture supernatant. As it is able to interact with any Fc of antibody, it will interefere with any WB detection whatever the tag. The easiest solution is to work with Staph strain deleted for Protein A encoding gene; the other easy solution is to eliminate protein A from the samples prior migration on SDS/PAGE by preclearing them with IgG acceptor beads.
This is a rare case in my experience. I would suggest you yo change another epitope tag for your protein (such as flag, myc, etc).
Hi there,
So obviously protein A is present in culture supernatant. As it is able to interact with any Fc of antibody, it will interefere with any WB detection whatever the tag. The easiest solution is to work with Staph strain deleted for Protein A encoding gene; the other easy solution is to eliminate protein A from the samples prior migration on SDS/PAGE by preclearing them with IgG acceptor beads.
Thank you for the answers. So I have about 2mls of my supernatant samples, where would I find a protocol for preventing the surface protein A with IgG acceptor beads?
Hi again,
I think IgG sepharose from GE will do depending on the amount of protein A you have in the sample (capacity of IgG sepharose is 2mg of protein A per mL of gel). For protein A fusion purification/elution instructions (it is not described for batch experiment) :
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314787424814/litdoc52208300AH_20110831144156.pdf
For your purpose, I think a basic CoIP protocol will do (mix sample with conditionated beads, incubation and then decantation).
Some additions to Dominique's suggestions:
If you already have Protein A or Protein A-Sepharose and amino reactive beads at your hands, you might simply want to extract IgG from some serum samples and then immobilize it yourself on NHS- or CNBr activated resin.
Furthermore, the resin for capturing Protein A may be regenerated by flushing with acidic buffer (pH 2 or so). Possibly saves time, money and resources. Works in batch and in column (syringe driven on 1ml devices) mode.
Another option might be using chicken antibodies (IgY) instead of IgG for detection in WB, especially if you can't get rid of ProteinA traces that still might interfere.
my 2c
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