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SDS PAGE Sample
i recently extracted proteins from a plant leaf, root and shoot and intended to analyze the proteins by SDS PAGE.
i prepared these samples by crushing the sample( i.e root, shoot, leaf) in extraction buffer, sterile distilled water and sample buffer. But i think i over diluted the samples. Once i ran SDS PAGE with these samples ( the maximum sample i can load is 30micro l)...i observed that the bands were tooo faint...and also there was a lot of trailing in lanes.
Could any 1 suggest ways to concentrate this already prepared sample so that i can get clear and distinct bands pleasee.
Re: SDS PAGE Sample
U can precipitate your proteins using common TCA/acetone precipitation protocol as below:
1. Add 1 volume of 100% TCA to 4 volumes of protein sample.
2. Incubate 10 min at 4°C.
3. Centrifuge at 14K rpm, 4°C for 10mins.
4. Remove supernatant and wash pellet with 200μl cold acetone.
5. Centrifuge at 14K rpm,4°C for 5mins.
6. Repeat steps 4-5 once again.
7. Air dry pellet and resuspend in appropriate buffer.
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