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SDS-Electrophoresis: General


Sodiumdodecylsulfate (SDS)

The sample treatment is “the half rent” within the SDS-electrophoresis.
Here you will find the major steps:

Mai 30, 2018

Native Protein

Sample Concentration
The protein concentration of the samples for a electrophoretic separation must be within a certain region.
Taking too less means nothing can be detected afterwards - and too much of protein overloads the separation leading too unsharp bands.
The protein concentration range depends on the sensitivity of the visualization process following the electrophoretic procedure:

The Coomassie Staining
This staining is less sensitive but can be quantificated.
Concentration range per slot with 15 ul sample volume: 1 - 5 ug

The Silver Staining
Very sensitive but no quantification possible.
Concentration per slot with 6 ul sample volume: 50 - 500 ng 

Sample Treatment and Marker
The SDS-molecule must first be added to the proteins. To achieve a stabil hydrophobic interaction between the protein and the tenside, the samples must be heated to 95° C.
nanogramm region (Coomassie-staining).
100 picogramm region (fluorescence)
picogramm region (silver-staining)
Standard: To control the sample concentration, the separation and the visualization process run a „Molecular Weight Marker“. After staining procedure, the samples should appear in the same state than this standard lane.
3.  After the samples dilution is done add 5% (v/v) Dithiothreitol (DTT)-solution (1% w/v) to the vials (reduction!). Do not reduce if the quarterny structure should be maintained!
4. Then heat 3 min at 95°C.
5. After the vials are cooled down add 5% (v/v) Iodoacetamide (IOA)-solution (4% w/v) to the samples (alkylation!). This fixes the reducing reaction's result on the proteins. Do not alkylate if you did not reduce the samples!

See the examples using IgG as target molecule on the right side.

The native protein...

After Cooking

...after heating to 95° with SDS...

After Reducing

The samples on a 10% gel.
Marker: Cambrex # 50547, 225kD - 5kD

Samples with the marker

...and after reducing

After Alkylation

The reduced groups are alkylated