I'm extracting total protein from rice leaf and would like to quantify the total protein. So I would like to ask if my protocol below is ok? I wrote my questions in between the steps:
Total protein extraction:
1) Prepare protein extraction buffer
(50mM Phosphate buffer pH 7, 0.1% SDS, 0.01M EDTA pH 8, 0.1% Triton X-100 and B-mercaptoethanol 10mM) and store in 4C.
- How long can i keep this buffer in 4 C?
- I prepare the phosphate buffer by mixing NA2HPO4 and NAH2PO4, is this ok?
2) Take out rice leaf sample from -80C and put in 2ml microcentrifuge tube (less than 100mg) (on liquid nitrogen). Add in a stainless steel bead and homogenize at 45Hz for 45s.
3) Add in 1ml protein extraction buffer per sample, invert to mix
4) Centrifuge 11000 rpm 10m 4C. Transfer supernatant to new tube
5) Centrifuge again 11000 rpm 10m 4C. Transfer supernatant to new tube
6) Aliquote protein 200ul per tube and store in -80C.
- how long can i keep the protein at -80C
Bradford assay
1) BSA standard
- I prepare stock at 1mg/ml BSA std in the protein extraction buffer as mentioned above. How long can i keep this stock in 4C?
2) Transfer 2, 4, 8, 12, 16, 20ul BSA std into microcentrifuge tube and top to 20ul. Blank was prepared with 20ul water. Thaw extracted protein samples and transfer 10ul into new tube, top to 20ul.
- Do i need to carry out this step on ice?
3) Add 980ul of Bradford reagent (5mg Coomassie Brilliant Blue, 5ml methanol, 10ml 85% Phosphoric acid, 850ml water, store at 4C) , invert mix, incubate at room temperature for at least 5 min (should I incubate on ice?).
- How long can I store the Bradford reagent in 4C?
4) Invert mix and transfer 200ul to microplate and read at 595nm within 30min.
Any suggestions and opinions are welcomed, thank you.
1. Do not add the reducing agent (mercaptoethanol) to the buffer until just before you are going to do the extraction because it is volatile and unstable to oxidation. Everything else should be OK to store at 4oC for a long time it is filter-sterilized. If it is not sterile, then freeze it. Mixing monobasic and dibasic sodium phosphate is a proper way to make the phosphate buffer.
6. You can probably keep the extract indefinitely at -80.
The detergents in your extraction buffer are going to be a problem for the Bradford assay because they interfere. If your sample is concentrated enough, you may be able to dilute it in water sufficiently to minimize the interference. The protein standards should be prepared under identical conditions to the samples, including the dilution of the detergent from the extraction buffer. The detergent concentration should be identical in all the standards, including the zero. I would not recommend storing the BSA solution at 4oC because of bacterial growth. Store it at -80oC, or -20oC for short-term.
2. You do not have to keep the standards on ice.
3. Commercially prepared Bradford reagent can be stored indefinitely at 4oC, but the home-made reagent may not be as stable. If you see precipitation in your dye, you should discard it.
4. It is not uncommon for the protein samples to precipitate when kept in the Bradford reagent for a long time (such as 30 min), because of the acidity. It is better to read after 5-10 min. Since you are going to use a plate reader to measure the absorbance, you might as well do the whole assay in the microplate. Use 10 microliter samples containing up to 10 micrograms of protein, in triplicate. Add 200 microliters of Bradford dye. Mix carefully to avoid spillage. Use a 96-well plate shaker if you have one, or the plate reader might have a mixing function. Read after 5-10 min.
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