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Sugar chain detection by chemical processing (TFMS processing) and Electrophoresis


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   Tools for sugar chain research!!
• Lectins
• GlyScope Series

The presence of the sugar chain can be found out by cutting off the sugar chain using acid and examining whether the molecular weight of the protein decreases. Here, by using trifluoromethanesulfonic acid, the molecular weight changes during acid treatment and can be examined by SDS-PAGE.

 

1. Equipment and apparatus.

Trifluoromethanesulfonic acid (TFMS)
Tris(hydroxymethyl)aminomethane (Tris)
Screw tube (with Teflon packing)
Thermostat with 0°C settings
Electrophoresis system for SDS-PAGE

 

2. Procedures.

  1. Add 10~50ug Sample protein into a screw tube, dry sufficiently.
  2. Add 50ul TFMS to dissolve the sample.
  3. Incubate at 0°C for 5 min to 1 hour.
  4. Add 500 ul 1M ice-cold Tris for neutralization.
  5. Dialyze against water.
  6. Apply SDS-PAGE, and compare the band positions after CBB staining.

 

3. Analysis example 1.

Figure 1. Molecular weight changes during TFMS processing.
Sample protein: Fetuin (from Fetal Calf Serum)

Leftmost: Molecular weight marker (From top, 97C66C45C29C20C14kDa)

1FWithout TFMS treatment
2FTreated by TFMS, 0°C, 5 min
3FTreated by TFMS, 0°C, 60 min

 

4. Judgement points.

The protein contains sugar chains, if the molecular weight decreases after TFMS treatment.

5. Attention.

*The appropriate time for TFMS treatment is different depending on proteins.
*If there is any water present, the reaction will be difficult to advance. TFMS is hygroscopic, thus please use new one.
*Since TFMS is a strong corrosive acid, please pay attention when using it. It will release fume in the air.

6. Option.

After treatment by TFMS, the absence of sugar chains can be examined not only by decrease of molecular weight, but also by lectin staining.
Instead of  CBB staining, perform lectin staining.
Once samples are treated by TFMS, performing multi-aspect analysis like this can increase the reliability and information of results.

6-1. Analysis example 2.

Figure 2. Lectin binding changes during TFMS processing.
Sample protein: Fetuin (from Fetal Calf Serum)
FFWithout TFMS treatment
1FTreated by TFMS, 0°C, 60 min
2FTreated by TFMS, 0°C, 4 hours
Lectin: Dojindo Labeling Phaseolus vulgaris (PHA-E4) lectin
Coloring: ABC reaction, Peroxidase

6-2. Judgement points.

After TFMS treatment, if the lectin staining has disappeared, as shown in figure 2, it can be considered that the protein contains sugar chains.

6-3. Attention.

*The appropriate time for TFMS treatment is different depending on proteins.
* The appropriate lectin is different depending on proteins.

7. Additional confirming experiments.

Confirm the question, [Do the sugar chains really disappear by TFMS treatment?] by hydrazinolysis, fluorescent labeling, and HPLC analysis.

Sample protein: Fetuin (from Fetal Calf Serum)

After hydrazinolysis, the sample was treated with ABOE labelling and Sialidase, and analyzed by reversed HPLC.

*Around 15 min (slash peak), there is N-type sugar chain, and around 35 min (grey peak), there is an O-type sugar chain. This experiment confirmed that the N-type sugar chains retains none of its original form after TFMS treatment (Figure 4).

*As shown in Figure 3 and Figure 4, it is suggested that there are easily broken sugar chains and difficultly broken ones  after TFMS treatment.

 

We sincerely appreciate the J-OIL MILLS Inc. for offering this manuscript.

   Tools for sugar chain research!!
• Lectins
• GlyScope Series

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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