SDS-Polyacrylamide Gel Electrophoresis at Neutral pH (NuPAGE)

Introduction and Principle

The traditional Laemmli system denaturing conditions are stimulated by the Invitrogen NuPAGE SDS-PAGE gel system which is a revolutionary high-performance polyacrylamide gel electrophoresis system. NuPAGE gels use a unique buffer formulation to maintain a neutral operating pH during electrophoresis, which minimizes protein modifications that can result in poor band resolution.

Gels are available in two formulations; Invitrogen NuPAGE Tris-acetate gels are ideal for separating large proteins while Invitrogen NuPAGE Bis-Tris gels are ideal for separating small to midsize proteins.  Further advantages are shorter separation time by using higher separation power and higher voltages.

Solutions/Reagents

• 39% acrylamide (w/v), 1% N, N’-methylene bisacrylamide (w/v)
  • Acrylamide and bisacrylamide (They are generally included to promote polymerization – [amazon link=”B00I31XTEM” link_icon=”amazon” /] or [amazon link=”B07F2ZZ1TL” link_icon=”amazon” /] )
• 86 M Bis-Tris (M_r 209.24), 1.71 M HCl (3.84 ml 37% HCl per 25 ml), adjusted to pH 6.5
  • Bis-Tris Buffer ( [amazon link=”B07329J6L8″ link_icon=”amazon” /])
• 10% TEMED (v/v) in ddH2O (stable at RT)
  • TEMED (A free radical stabilizer, used as a catalyst for polyacrylamide gel electrophoresis – [amazon link=”B00I31VPQQ” link_icon=”amazon” /] )
  • Distilled Water (Used in the dilution of reagents – [amazon link=”B07MFS5Z3L” link_icon=”amazon” /])
• 10% ammonium persulfate (w/v) in ddH2O (stabilize for 2–3 days at 4 ◦C)
  • Ammonium Persulfate [APS] (Used as a catalyst for acrylamide gel polymerization – [amazon link=”B01N4KDS0X” link_icon=”amazon” /])
• MES electrophoresis buffer 20-fold: 1 M MES (Mr 195.2), 1 M Tris, 2% SDS (w/v), 0.6% EDTA (free acid) (w/v), pH 7.3
  • MES SDS Running Buffer (It is preferred for separating small- to medium-sized proteins – [amazon link=”B07D7GWDJS” link_icon=”amazon” /])
• MOPS electrophoresis buffer 20-fold: 1 M MOPS (Mr 209.3), 1 M Tris, 2% SDS (w/v), 0.6% EDTA (free acid) (w/v), pH 7.7
  • MOPS Buffer (Used for separating small- to medium-sized proteins, provides increased resolution of small MW proteins, and shortens the electrophoresis run time – [amazon link=”B073ZJSLTZ” link_icon=”amazon” /])
• sample buffer fourfold: 0.986 M Tris, 40% sucrose (w/v), 8% SDS (w/v), 0.06% EDTA (w/v), 0.075% (w/v) Coomassie Brilliant Blue R250, 0.25%) phenol red (w/v)
  • Tris (Used in the buffer solution -[amazon link=”B07D85GJPN” link_icon=”amazon” /] )
  • Sucrose (It makes the sample denser, so the sample will remain in the bottom rather than floating out – [amazon link=”B005TLIA9I” link_icon=”amazon” /] )
  • Sodium Dodecyl Sulfate (It is a detergent that is used to denature proteins – [amazon link=”B07F2PP914″ link_icon=”amazon” /])
  • Ethylenediaminetetraacetic acid (EDTA) (Water-soluble solid used to prepare solutions – [amazon link=”B07MJDDPQK” link_icon=”amazon” /] )
  • Coomassie Brilliant Blue R-250 (It binds proteins – [amazon link=”B07MLYTNJD” link_icon=”amazon” /])
  • Phenol Red (Used as a pH indicator – [amazon link=”B077T2PQLB” link_icon=”amazon” /] )
• blotting buffer20-fold: 0.5 MBicine (N,N-bis(2-hydroxyethyl)- glycine), 0.5 M Bis-Tris, 20 mM EDTA
  • Blotting buffer (Used to decrease the migration of the protein out of the gel – [amazon link=”B01NCS7H2I” link_icon=”amazon” /])

Preparation of reagents and Experiment Protocol

Step 1:

Mix the gel according to the given instructions and pour it into the cassette. Put the comb directly into the separation gel since a stacking gel is not necessary. The gel is ready for use after the complete polymerization.

Step 2:

Now mount the gel into the electrophoresis apparatus; fill the electrode chambers with 1:20 diluted MES or MOPS electrode buffer. A concentration gradient gel with 4 → The separation of MES buffer with 12%T running is between 2 and 250 kD; while performing with MOPS buffer, the separation range is between 10 and 250 kD.

Step 3:

With one-third of their volume, mix the samples and heat at about 70 ◦C for 5 min.

Step 4:

To a final concentration of 10 mM add DTT for cleavage of disulfide bridges. For at least 50– 60 minutes run a gel of 8 cm separation length with 200 V cv. To carry off the heat surround the gel completely with electrode buffer during electrophoresis.

Step 5:

After electrophoresis, the gel is stained and fixed as other PAGE gels, too. After electrophoresis, the gel is stained and fixed as other PAGE gels, too. The 1:20 diluted buffer H is recommended for semi-dry blotting.

Comments/Conclusions:

The NuPAGE gel system comes in two Bis-Tris gel concentrations (10% or 4-12%) in 10-, 12-, 15-, or in 1 well or 2-D well format. In addition, the two gel types can be combined with two SDS running buffers (MES-SDS and MOPS-SDS) to provide four separation ranges between 1.5 and 250 kDa. Also available is the NuPAGE Transfer Buffer, which maintains a neutral environment throughout the transfer, improving blotting efficiency and Antioxidant additive, and NuPAGE Sample Buffer, which maintains complete sample reduction throughout the run.

Introduction and Principle: SDS-Polyacrylamide Gel Electrophoresis According to WEBER, PRINGLE, and OSBORN

It describes methods for the characterization of proteins separated on SDS gels and sodium dodecyl sulfate (SDS) gel electrophoresis. Proteins are disassociated by SDS into their constituent polypeptide chains. Polypeptide chains are separated according to their molecular weights in the presence of SDS in polyacrylamide gel electrophoresis. Thus, by comparing the proteins’ electrophoretic mobilities on SDS gels to the mobilities of marker proteins, the length of the polypeptide chains of a given protein can be determined with well-characterized polypeptide chain molecular weights. SDS-polyacrylamide gel electrophoresis is rapidly and easily performed, can be used with microgram amounts of protein, and, requires only inexpensive equipment.

With respect to pH, this PAGE is a continuous system, and the resolution is minimum than that of a disc system. The convenience lies in the use of buffers free of primary amino groups; therefore, an electrotransfer is intended because a buffer changes separation performance (broadening of bands by diffusion during buffer change) and decreases transfer yield. The technique is both reproducible and reliable, and the results are easy to interpret.

Solutions/Reagents

• 4% acrylamide (w/v), 1.2% N, N’ Solutions/Reagents -methylene bisacrylamide (w/v) in ddH2O (%C = 2.65)
  • Acrylamide and bisacrylamide (They are generally included to promote polymerization – [amazon link=”B00I31XTEM” link_icon=”amazon” /] or [amazon link=”B07F2ZZ1TL” link_icon=”amazon” /])
  • Distilled Water (Used in the dilution of reagents – [amazon link=”B07MFS5Z3L” link_icon=”amazon” /] )
• 2 M sodium phosphate buffer, pH 7.2, 0.2% SDS (w/v) (do not use potassium phosphate) (7.8 g NaH2PO4 · H2O, 38.6 g Na2HPO4 · 7H2O and 2 g SDS in 1000 ml ddH2O)
  • Sodium Phosphate buffer (Used to provide ions that carry a current and to maintain the pH at a relatively constant value – [amazon link=”B0732CNYNK” link_icon=”amazon” /] )
  • Sodium Dodecyl Sulfate (It is a detergent that is used to denature proteins – [amazon link=”B07F2PP914″ link_icon=”amazon” /])
• TEMED (v/v) in ddH2O (stable at RT)
  • TEMED (A free radical stabilizer, used as a catalyst for polyacrylamide gel electrophoresis – [amazon link=”B00I31VPQQ” link_icon=”amazon” /])
• 10% ammonium persulfate (w/v) in ddH2O (stable for 2– 3 days at 4 ◦C)
  • Ammonium Persulfate [APS] (Used as a catalyst for acrylamide gel polymerization – [amazon link=”B01N4KDS0X” link_icon=”amazon” /])
• sample buffer: 10 mM sodium phosphate buffer, pH 7.2, 2% SDS (w/v), 4% 2-mercaptoethanol (v/v)

Preparation of reagents and Experiment Protocol

Step 1:

Pipette the solutions for preparing the separation gel and adjust the required volume with ddH2O, and mix well (avoid foam-forming).

Step 2:

With the addition of Soln. D starts the polymerization reaction. Introduce the mixture into the cassette about 10 mm from the top of the front plate without air bubbles and gently layer ddH2O or n-butanol to keep a smooth surface. Polymerization needs 20–30 minutes and maybe prolonged by decreasing or cooling the amount of Soln. D, and vice versa.

Step 3:

By the use of filter paper, carefully remove the liquid above the gel just before performing the electrophoresis and prepare the stacking gel. Pour the stacking gel immediately after introducing the separation gel instead of covering with ddH2O or n-butanol since it is a continuous separation system but getting sharp separations between both gels is a somewhat sophisticated process. 

Step 4:

With a protein concentration, of no more than 20 mg/ml prepare the sample in buffer E. For about 2–3 minutes heat the solution to 95 ◦C and supplement with a droplet of glycerol or some crystals of sucrose or 1/10 volume of bromophenol blue in 50% sucrose solution.

Step 5:

With an equal volume of double concentrated sample buffer E, mix the dissolved sample and electrode buffer is Soln. B, diluted 1:1 with ddH2O.

Comments/Conclusions

As this system is susceptible to ions; therefore, if the sample has an (expected) ionic strength > 0.05, it has to be dialyzed against the sample buffer. As a principle, for Coomassie staining at least 1 µg of protein per band is needed.

Voltage regime and fixation (method and results calculation) are the same as for the Laemmli system (as discusses in the previous article), staining as usual.

Introduction and Principle: Urea-SDS-Polyacrylamide Gel Electrophoresis for the Separation of Low Molecular Weight Proteins

High concentration gels are needed in order to analyze low molecular weight proteins by this electrophoresis system, but these gels are very frail and thus hard to handle. With a change in the basic Laemmli SDS-PAGE protocol, proteins with 10 kDa or less will be separated maintaining a good resolution with reproducible results. The allowance of separation is due to the high concentration of acrylamide (%T = 13.35, %C = 6.22), as well as the presence of urea, especially of low molecular weight polypeptides and proteins.

Solutions/Reagents:

• 6% TEMED (v/v), 0.8% SDS (w/v), 0.8 M phosphoric acid, adjusted with solid Tris to pH 6.8
  • TEMED (A free radical stabilizer, used as a catalyst for polyacrylamide gel electrophoresis – [amazon link=”B00I31VPQQ” link_icon=”amazon” /])
  • Sodium Dodecyl Sulfate (It is a detergent that is used to denature proteins -[amazon link=”B07F2PP914″ link_icon=”amazon” /] )
  • Phosphoric Acid (Used to make buffer – [amazon link=”B010199QIQ” link_icon=”amazon” /] )
  • Tris pH 6.8 (Used in buffer solution – [amazon link=”B079PZVZDD” link_icon=”amazon” /] )
• 5% acrylamide (w/v), 1.25% N, N’-methylene bisacrylamide (w/v) in ddH2O
  • Acrylamide and bisacrylamide (They are generally included to promote polymerization – [amazon link=”B00I31XTEM” link_icon=”amazon” /] or [amazon link=”B07F2ZZ1TL” link_icon=”amazon” /])
  • Distilled Water (Used in the dilution of reagents – [amazon link=”B07MFS5Z3L” link_icon=”amazon” /] )
• 85 M urea, 50 mM phosphoric acid, 2% SDS (w/v), 1% 2- mercaptoethanol (v/v), 0.1 mM EDTA, adjusted with solid Tris to pH 6.8
  • Urea (Denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight – [amazon link=”B01BCSBPGG” link_icon=”amazon” /] )
  • Phosphoric Acid (Used to make buffer – [amazon link=”B010199QIQ” link_icon=”amazon” /])
  • Sodium Dodecyl Sulfate (It is a detergent that is used to denature proteins – [amazon link=”B07F2PP914″ link_icon=”amazon” /])
  • Ethylenediaminetetraacetic acid (EDTA) (Water-soluble solid used to prepare solutions – [amazon link=”B07MJDDPQK” link_icon=”amazon” /] )
  • Tris pH 6.8 (Used in buffer solution – [amazon link=”B079PZVZDD” link_icon=”amazon” /] )
• electrode buffer: 0.1 M phosphoric acid, 0.1% SDS (w/v), adjusted with solid Tris to pH 6.8

Preparation of reagents and Experiment Protocol

Step 1:

Prepare solution A’ by dissolving 1 mg of ammonium persulfate in 1 ml Soln. A, before pouring the gel.

Step 2:

Prepare a fresh sample of solution B’ by dissolving 0.125 g N, N’-methylene bisacrylamide in 10 ml of Soln. B (do not heat above 30 ◦C) and 4.114 g urea.

Step 3:

Mix the gel gently according to A’ (1.25 ml/10 ml), B’ (4.70 ml/10 ml) and C’ (4.05 ml/10 ml) and introduce it into the cassette without delay. Into the liquid polyacrylamide mixture, insert the comb so that polymerization proceeds at room temperature.

Step 4:

Mix samples with an equal volume of Soln. C.

Step 5:

Perform separation with 3 V/cm at RT overnight, or 6–8 V/cm (with cooling) and with electrode buffer D fill the electrophoresis chambers. The whole gel length is not used for separation, but to stop the run if the tracking dye has moved three-quarters of the gel.

Comments/Conclusions

To calculate the results, fix the gel thoroughly either in TCA or 5-sulfosalicylic acid (as discussed in the previous article) after electrophoresis to remove urea prior to staining. Once urea is calculated, take the readings accordingly.

References

1. Swank, RT., Munkres, KD. (1971). Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem; 39(2):462-77.
2. Weber, K., Pringle, JR., Osborn, M. (1972). Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol; 26:3-27.
3. Kathrin, Kusch., Marina, Uecker., Thomas, Liepold., Wiebke, Möbius., Christian, Hoffmann., Heinz, Neumann., Hauke, B. Werner., and Olaf, Jahn. (2017). Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome; 5(1): 3.
4. Rudi, Hrncic., Jonathan, Wall., Dennis, A. Wolfenbarger., Charles, L. Murphy., Maria, Schell., Deborah, T. Weiss., and Alan, Solomon. (2000). Antibody-Mediated Resolution of Light Chain-Associated Amyloid Deposits. Am J Pathol; 157(4): 1239–1246.