EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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complex samples

Mai 30, 2018

Complex samples such as E.coli dry powder - plant tissues - etc. tend to agglutinate during the stacking process due to the SDS-fat mycelles.
After this false starting process the sample form a “channel” in the track.
Remedy:
1. Use a extraction buffer without SDS.
This inhibits the solubilization of fat. Afterwards the fat-SDS-mycelles will disturb the electrophoresis.
The SDS-samples should be produced out of this concentrate.
2. Add 0.6 M Urea to the samples after the heating process.


ad 1: Extraction Buffer (100 ml)

250 mM TRIS

3.04g

190 mM Glycine

1.5 g

1 mM EDTA

30 mg

0.01% Dodecylmaltoside

10 mg

0.005% Triton X100 (1%)

500 ul

(add freshly: 3 M Urea)

(180 mg / 1 ml)

Fill up to 100 ml. The pH-value should be ~9.2

ad 1: extraction procedure:

Ultrasonic treatment

15 min

Centrifuge 12000 rpm

5 min

Then the SDS-sample procedure should follow


ad 2: adding urea

after the SDS-sample procedure:
After the samples cooled down, add 3 M Urea (180 mg/1 ml) and shake well


TS_ComplexMix
Pfeil

sample #4 is E.coli directly prepared with SDS sample-buffer.

EColiExtract3MUrea

... samples #3-7 is E.coli prepared with extraction buffer