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SDS PAGE Sample

SDS PAGE Sample - Molecular Biology Techniques

SDS PAGE Sample - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 07-07-2011, 03:22 PM
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Default SDS PAGE Sample



Dear all,
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.
THNX
NIS
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Old 08-06-2011, 04:43 AM
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Default 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 4C.
3. Centrifuge at 14K rpm, 4C for 10mins.
4. Remove supernatant and wash pellet with 200μl cold acetone.
5. Centrifuge at 14K rpm,4C for 5mins.
6. Repeat steps 4-5 once again.
7. Air dry pellet and resuspend in appropriate buffer.
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