I would like to learn about methodologies for protein extraction from microalgae (Scenedesmus and Chlorella) using the smallest amount of biomass as possible for a western blot assay.
This is a question you can only answer experimentally, there are several factors here
1. the level of expression of the protein of interest will have a bearing on the minimal amount of starting material
2. the efficiency of the antibody you plan yo use for its epitope will determine how sensitive the assay is
The detection system (colourmetric, chemiluminescence or fluorescence) will also have an impact on what you are going to be able to detect in terms of protein concentration
I would recommend you TCA protein extraction. With this method you can concentrate your total protein to the amount you want.
I find your question too general type, because any way you need very small amount of protein in Western blot assay, for the simple reason that the gel must show separated bands without which you can not run Western blot assay. Kindly give some more details so that you may get a better answer.
I have around 20 mg of biomass to use as I'm using 10 mL cultures of transfected microalgae, the problem is I'm using a plasmid with a CaMV35s promoter (no induction) so i dunno if I can get enough of my protein.
I have thought about the TCA methodology but I have never used it before so I would like to get some info about this protocol.
I tried using glass beads for DNA extraction and It worked quite well with 20 mg of wet biomass, so I was hoping it would be also enough for the protein extraction.
Thanks!
Hi Alejandro, here is protocol for protein extraction by TCA:
1. Resuspend your cells in 200 mcl of 10 % TCA. Add ~250-300 mcl of glass beads (0.5-1 mm); vortex extensively for 15-20 min.
2. Add 800 mcl of 10 % TCA, mix, take extract to fresh eppi.
3. Spin at 3000 rpm for 10 min. Remove supernatant completely.
4. Add 100-200 mcl of 1x Laemmli sample buffer (it will turn yellow), then add 50-100 mcl of 1M tris-base to neutralize (samples will turn blue again). Resuspend pellet.
5. Boil for 10 min. Spin for 10 min at full speed in microfuge.
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