SDS TS Fat

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Too much fat in the sample

April 28, 2023


	Precipitating material at the anodal slot-edge, channal forming (See Track 2)




		1) Too much fat in the sample

	Remedy #1: Use special extraction buffer


	Do not extract your proteins directly from the raw materials using detergent (SDS) -containing buffers! Correct extraction procedure: Extraction buffer: 0.25 M TRIS, 0.2M Glycine, 0.001 M EDTA (pH 9.1), detergent max. 0.05% Extraction procedure: 15 min ultra sonic treatment - 10 min centrifugation 10 000 rpm - take the supernatant!


	Remedy #2: Defatting the samples with Kerosene (simple procedure), without sample concentration


	Add (~10% of the sample volume) Kerosene (stained with Sudan-red) and shake well. Leave 5 min to separate the Kerosene from the aqueous phase. Do not take the red hydrophobic phase. Then add SDS, the reducing agent (DTT) and heat 3 - 5 minutes to 95 °C

		Remedy #3: Acetone-precipitation with optional sample-concentrating

			Precipitate the proteins with 100% Acetone. Cooling, then centrifuge samples. Resolubilize with sample buffer, + heating 3-5 min 95°.

			Defatting and desalting with optional concentration of the samples

Action	Remarks	Time
precipitation with 100% Acetone	50 µl sample + 200 µl 100% Acetone	---
cooling at -20°C	Cool down to precipitate completely at (-20°C)	60 min
centrifugation	15 000 rpm, then discard the supernatant	10 min
drying	open the cups to remove the Acetone	30 min
resuspensation / concentration	add (fe.) 10 - 100 µl sample buffer (2% SDS)	---
heating the samples	95°C: will charge the proteins with SDS	3-5 min


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