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Conductscience Administrator
Conduct Science promotes new generations of tools for science tech transferred from academic institutions including mazes, digital health apps, virtual reality and drones for science. Our news promotes the best new methodologies in science.
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  • SDS-Polyacrylamide Gel Electrophoresis at Neutral pH (NuPAGE)
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Introduction and Principle

Proteins in the mass range 1–100 kDa are commonly separated by Tricine–SDS-PAGE. For the resolution of proteins less than 30 kDa, it is the preferred electrophoretic system. As compared to other electrophoretic systems, the concentrations of acrylamide used in the gels are lower. Electroblotting is facilitated by these lower concentrations, which is particularly crucial for hydrophobic proteins. Tricine–SDS-PAGE is also used for doubled SDS-PAGE (dSDS-PAGE), and it offers advantages for resolution of the second dimension after clear-native PAGE (CN-PAGE) and blue-native PAGE (BN-PAGE). Doubled SDS-PAGE (dSDS-PAGE) is a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification.

Solutions/Reagents:

  • 48% acrylamide (w/v), 1.5% N,N’-methylene bisacrylamide (w/v)
    • Acrylamide and bisacrylamide (They are generally included to promote polymerization – )

A’ – 46.5% acrylamide (w/v), 3.0% N,N’-methylene bisacrylamide (w/v)

  • 3 M Tris, adjusted with HCl to pH 8.45, 0.3% SDS (w/v)
  • 10% TEMED (v/v) in ddH2O
    • TEMED (A free radical stabilizer, used as a catalyst for polyacrylamide gel electrophoresis – )
    • Distilled Water (Used in the dilution of reagents – )
  • 10% ammonium persulfate (w/v)
    • Ammonium Persulfate [APS] (Used as a catalyst for acrylamide gel polymerization – )
  • anode buffer: pH 8.9, 0.2 M Tris-HCl
    • Anode buffer (It is used to separate proteins in the mass range 1–100 kDa – )
  • cathode buffer: 0.1 M Tricine (Mr 179.2), pH 8.25, 0.1 M Tris, 0.1% SDS (w/v)
    • Cathode buffer (It is used to separate proteins in the mass range 1–100 kDa – )
  • sample buffer fourfold: 12% SDS (w/w), 150 mM Tris, 50 mM DTE or DTT, 30% glycerol (v/v), 0.05% (w/v) Coomassie Brilliant Blue G250, pH 7.0

Preparation of reagents and Experiment Protocol

Step 1:

Prepare the stacking gel and separation gel as given. The stacking gel can be carefully poured directly onto the fresh (liquid) separation gel, and the separation gel may be overlaid with ddH2O and poured separately.

Note:
It is suggested to use a separation gel between separation and stacking gel for gels with %T > 10 and %C > 3. (Length of separation should be about 1/5 to 1/10 of the total gel length).

Step 2:

Dissolve the samples in buffer G (2–5 µg peptides or 0.5–2 µg protein per expected band, depending on the detection method). Mix liquid samples in a ratio of 1 vol. of buffer (fourfold) to 3 vol. of sample, dissolve solid samples in 1:4 diluted buffers G, for approximately 9-10 minutes heat to 80 ◦C, and add the samples to form a layer below the cathode buffer in the sample pockets of the gel.

Note:

Coomassie Brilliant Blue G-250 is recommended as a tracking dye. Bromophenol blue does not work well since it moves significantly slower than small peptides.

Step 3:

Run the electrophoresis at room temperature, i.e., 8-11, 6-7, 6.5-7.5 V/cm gel length.

Comments/Conclusion:

After the run, common staining and fixation protocols are used to calculate the results.

Introduction and Principle: SDS-Polyacrylamide Gel Electrophoresis at pH 2.4

This method allows the separation of alkali-labile proteins (e.g., acylphosphate phosphoproteins) under denaturating conditions according to their molar mass. Despite the low acrylamide concentration (%T = 5.61, %C = 3.61), the separation force is remarkable. Because it is an SDS-containing system, the migration is from “−” to “+” despite the low pH.

Solutions/Reagents:

  • 0% acrylamide (w/v), 1.5% N,N’ -methylene bisacrylamide in ddH2O
    • Acrylamide and bisacrylamide (They are generally included to promote polymerization – )
  • 1 M sodium phosphate-phosphoric acid buffer, pH 2.4
  • 20% SDS (w/v) in ddH2O
    • Sodium dodecyl sulfate (SDS) 20% (It is a detergent that is used to denature proteins – )
    • Distilled water (Used in the dilution of reagents – )
  • 5 mM ascorbic acid
    • Ascorbic Acid (Promotes polymerization – )
  • 025% Fe(II)SO4 · 7H2O (w/v) in ddH2O
    • Ferrous Sulphate (It provides iron for the synthesis of iron-containing proteins – )
  • 5% H2O2
    • Hydrogen Peroxide 2.5% (Used for electrophoretic analysis of RNA – )
  • sample buffer: 50 mM sodium phosphate buffer, pH 2.4, 2% SDS (w/v), 2% 2-mercaptoethanol (v/v) or DTT
  • electrode buffer: 50 mM sodium phosphate buffer, pH 2.4, 0.1% SDS

Preparation of reagents and Experiment Protocol

Step 1:

Solution Gel (ml/10 ml) Buffer I (ml/10 ml)
A 1.40
B 0.50
C 0.50 0.05
D 1.00 1.00
E 0.10 (Add H2O to 9.99 ml) 0.10 (Add H2O to 9.99 ml)
F 0.01 0.01

Mix the gel according to the table below, and after pouring the mixture into the cassette cover it with Soln. I (check the table below). The gel should be prepared 36–48 hours before the electrophoresis as the polymerization takes place very slowly. There is no need for a stacking gel.

Step 2:

Now, dissolve the samples in buffer G. Apply voltage up to 8–10 V per centimeter of gel length to the electrode buffer Soln H.

Comments/Conclusion:

After applying the voltage, the tracking dye has to be an anionic one completely colored at pH 2.4 (e.g., Pyronin Y or cresol red).

Introduction and Principle: Urea-Polyacrylamide Gel Electrophoresis for Basic Proteins at pH 2

Described by Panyim and Chalkley in 1969, it is a continuous acetic acid-urea (AU) gel system that could separate very similar basic proteins based on effective charge and differences in size. For example, unmodified histone H4 can be separated from its monophosphorylated or monoacetylated forms. The major determinant of electrophoretic mobility is basic proteins with a high isoelectric point that will clearly have a net positive charge at the acidic pH 3 of this gel system. A significant decrease in effective gel mobility is observed if a single of these positive charges is removed. Likewise, the net positive charge of the protein during gel electrophoresis by one is decreased by the addition of a phosphate moiety. Additive Triton X100, a non-ionic detergent, may increase the resolution.

Solutions/Reagents:

  • 0% acrylamide (w/v), 0.4% N,N’-methylene bisacrylamide (w/v) in ddH2O
    • Acrylamide/Bis Solution, 30% (They are generally included to promote polymerization – ) – Use two bottles for 60%
    • Distilled water (Used in the dilution of reagents – )
  • 5% acetic acid (w/v), 5.4% TEMED (v/v) in ddH2O
    • Acetic Acid (Acts as the base for effective protein separation – )
    • TEMED (A free radical stabilizer, used as a catalyst for polyacrylamide gel electrophoresis – )
  • 4 M urea in ddH2O
    • Urea (Denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight – )
  • electrode buffer:5 M urea, 0.9 M acetic acid

Preparation of Reagents and Experiment Protocol:

Step 1:

Urea and acrylamide must be ultra-pure. Do not allow to warm their aqueous solutions above 35 ◦C. The gel resulting from the table below has %C = 0.66 and %T = 15.1. There is no need for a stacking gel.

Solution (ml/10 ml)
A 2.50
B 1.25
C 6.25
Ammonium persulfate (added as solid) 12.5 mg

Step 2:

A pre-electrophoresis is essential prior to protein separation and is done overnight with 3.5 mA/cm2 (constant current). After pre-electrophoresis, the electrode buffer D is renewed. Dissolve the samples in buffers C or D, supplemented with some crystals of sucrose.

Comments/Conclusion:

Run the electrophoresis at 8–10 V/cm (cv) so the proteins migrate from “−” to “+.” By neutral red, the electrophoresis font may be indicated.

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