EDC Impressum & DSE

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)

First Trial

2) Too much salt / buffer in the sample

Remedy #1: chromatography-desalting without concentrating

Mai 30, 2018

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

Second Trial

Fig.2: After adding urea to the samples

Adding Urea

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.

NAP-5

Fig.5: Desalting sample with a NAP-5

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

Sample 5!

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.

Clean 15%

Fig.6: Samples in a 15% gel

Under Construction!

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 Ál sample + 50 Ál 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 Ál 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 Ál sample + 150 Ál 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 Ál 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]