SDS TS Salt

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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		SDS: Too much Salt / Buffer in the Sample

	Unsharp bands, lateral band broadening (See Track 1,2,3)



			2) Too much salt / buffer in the sample

Remedy #1: chromatography-desalting without concentrating

April 28, 2023


	First trial: After the samples were filled in the sample-wells and the voltage is applied sample 1 – 5 jumped out of their slots and run over the stacking gel. This osmotic effect happens when there are too much ions in the sample volumes. It can also happen that 'salty samples' leave their slots even without an applied electric field. See figure 1.


	Second trial: To overcome this trouble, 1 spatula urea is added ( 3 – 4M) to 100 ul of the SDS-samples, see figure 3 . The effect of this procedure: Samples 1 – 5 stay in their slots. Adding of urea in slot 5 was not sufficient. This sample migrates very slow out of the slot, shrinking to a slim line. See fig. 2




	*Fig.2:* After adding urea to the samples




	*Fig.3:* Adding urea to the samples

Special treatment for sample 5: This sample is passed through a special desalting column (NAP-5 GE 17-0853-01). This Sephadex G-25 containing ready-to-use column is especially designed to desalt 500ul DNA-samples. For proteins the procedure is changed a little: See table 1 and figure 5 and note that there is no sample dilution!
Figure 4 shows the proper run of the sample 5 together with the other samples.




		*Fig.5:* Desalting sample with a NAP-5

	*Table 1:* NAP-5 desalting protocol for proteins (and DNA)




	*Fig.4:* Sample 5 after NAP-5 treatment

NAP-5 Prot.	Equilibration	Sample Load	After sample	Elution
DNA	3 x 3 ml	500 ul	-----	1 ml
Protein 500 ul	3 x 3 ml	500 ul	500 ul	500 ul
Protein 100 ul	3 x 3 ml	100 ul	750 ul	400 ul


	Last optimization: The gel concentration. All samples show an anodal cut because they were migrating too fast. A 15%T-gel is compared with a 12.5%T-gel, see figure 6 and 7.




	*Fig.6:* Samples in a 15% gel




	*Fig.7:* Samples in a 12.5% gel


		Remedy #2: Trichloroacetic Acid (TCA) -precipitation with optional sample-concentrating

		Precipitate the proteins with 20% Trichloric Acid. Centrifuge samples. Wash 1 x with dist.water. Centrifuge. Resolubilize with sample buffer, + heating 3-5 min 95°. Additional advantage beside the desalting effect: sample concentrations can be increased easily!

	Desalting (concentration) the samples

Action	Remarks	Time
precipitation with 20% TCA	fe. 50 µl sample + 50 µl 20% TCA	---
cooling on ice	chill down the precipitate on ice (0°C)	30 min
centrifugation	15 000 rpm, then discard the supernatant	10 min
washing	purge the pellet carefully with water to remove the TCA	~10 sec
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


		Remedy #3: Ethanol (EtOH) -precipitation and defatting with optional sample-concentrating

	Desalting (concentration) the samples

Action	Remarks	Time
precipitation with 67% EtOH	fe. 50 µl sample + 150 µl 20% TCA	---
cooling at 4°C	cool down the precipitate in a fridge (4°C)	60 min
centrifugation	15 000 rpm, then discard the supernatant	10 min
drying	open Eppendorf cup to remove EtOH (res.vacuum)	10-20 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


	Remedy #4: Aceton-precipitation and defatting with optional sample-concentrating:


	[fat in the samples]


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