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Protein Electrophoresis & Analysis

Protein Loading Buffer Pack

Product information

Applications

• Preparation proteins for SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Description

The Protein Loading Buffer Pack is used for preparation of protein samples for denaturing SDS-polyacrylamide gel electrophoresis. This buffer contains all the necessary reagents for complete disruption of high-order protein structures. The SDS included in the buffer binds to hydrophobic regions of the protein, causing the protein to unfold and giving it a negative charge. DTT breaks disulfide bonds and destroys residual secondary structures. As DTT is prone to oxidation during multiple freeze-thaw cycles it is supplied in separate vials. Glycerol is included in the loading buffer to ensure protein samples remain in gel wells after loading. The bromophenol blue dye allows for visualization of probes and monitoring the progress of electrophoresis.

Quality Control

Tested in protein sample preparation prior to SDS-PAGE.

Components

• 5X Loading Buffer: 0.313 M Tris-HCl (pH 6.8 at 25°C), 10% SDS, 0.05% bromophenol blue and 50% glycerol.
• 20X Reducing Agent: 2 M DTT.

Storage

5X Protein Loading Buffer store at -20°C or at 4°C.
20X Reducing Agent store at -20°C.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Protein Samples: Extraction and Quantification Guides

Sample/procedure	Extraction	Quantification
Mammalian cell and tissue samples	Extract total proteins using ProteoJET™ Mammalian Cell Lysis Reagent. Extract nuclear and cytoplasmic proteins using ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit.	Use Bradford Reagent, ready-to-use for protein quantification. Easily create standard curves by using one of our convenient protein standard sets: Bovine Serum Albumin Standard Set, Bovine Gamma Globulin Standard Set, Protein Standard Solution. Create a standard curve by plotting the 595 nm values (y-axis) versus their concentration in µg/ml (x-axis). Use the standard curve to determine the protein concentration of each unknown sample.
Bacterial samples	To analyze total bacterial proteins in SDS-PAGE, cells can be treated directly with DualColor™ Protein Loading Buffer Pack or Protein Loading Buffer Pack.
Lyophilized proteins	To analyze proteins in SDS-PAGE, treat lyophilized proteins directly with DualColor™ Protein Loading Buffer Pack or Protein Loading Buffer Pack.

Protein Samples: Preparation for Loading on SDS-PAGE

Step	Sample preparation with the DualColor™ Protein Loading Buffer Pack	Sample preparation with the Protein Loading Buffer Pack
Thaw	Thaw the buffer pack components either at room temperature or at 37°C for a few minutes to dissolve precipitates.
Mix	Vortex gently, but thoroughly to ensure that the solution is homogeneous.
Dilution	2.0 µl of 20X Reducing Agent	2.5 µl of 20X Reducing Agent
	Protein sample (~0.5 ng – 2.5 µg) For Western blots or gels to be treated with Coomassie based stains, use up to 2.5 µg of total protein per minigel well. For silver staining applications use up to 10 ng of total protein per minigel well.
	10 µl of 4X DualColor™ Protein Loading Buffer Water, nuclease-free to 40 µl*	10 µl of 5X Protein Loading Buffer Water, nuclease-free to 50 µl*
Denaturation	Incubate samples at 95-100°C for 5 minutes.
Loading	Spin for a few seconds in a microcentrifuge. Apply directly to an SDS-polyacrylamide gel. Use ~10 µl per minigel well.

* The sample volume can be scaled up or down.

Semi-dry Protein Transfer for Western Blotting

Note

Wear gloves throughout the procedure to avoid contamination. Use 100 ml of each solution for mini gels (8x10 cm; 10x10 cm), for larger gels use enough of the solution to completely cover the gel/membrane/paper sheets in each step.
Buffers
Tris-glycine-methanol protein transfer buffer. Cool at 4°C before use.

Component	Amount	Final concentration
10X Tris-glycine buffer	10 ml	1X
Methanol	10 ml	10% (v/v)
Deionized water	to 100 ml

CAPS buffer for electrotransfer of proteins onto PVDF for N-terminal sequencing. Cool at 4°C before use.

Component	Amount	Final concentration
10X CAPS (100 mM, pH 11.0)	10 ml	10 mM
Methanol	10 ml	10% (v/v)
Deionized water	to 100 ml

10X CAPS (3-[cyclohexylamino]-1-propanesulfonic acid). Store at 4°C.

Component	Amount	Final concentration
CAPS	2.21 g	100 mM
Deionized water	to 90 ml
2N NaOH	titrate to pH 11.0 (~4 ml)
Deionized water	to 100 ml

Ponceau S staining solution (only freshly made staining solution should be used).

Component	Amount	Final concentration
Ponceau S	0.2 g	0.2% (w/v)
Glacial acetic acid	1 ml	1% (v/v)
Deionized water	to 100 ml

Semi-dry Protein Transfer

1. Presoak 2-4 pieces of blotting paper (cut to the size of the gel) in transfer buffer for 5 min.
2. Cut a piece of nitrocellulose membrane to the size of the gel and equilibrate it in transfer buffer. If a PVDF membrane is used, incubate it in methanol for 2 min before equilibrating it in transfer buffer. Use CAPS buffer for N-terminal sequencing, Tris-glycine-methanol protein transfer buffer is suitable for all other applications.
3. Carefully remove the stacking gel from the resolving gel. Soak the resolving gel in CAPS buffer for 5 min if this buffer is used. This step can be omitted if Tris-glycine-methanol protein transfer buffer is used.
4. Assemble the transfer sandwich with the resolving gel on the anode (+) as shown in Figure below. Use one sheet of blotting paper or two pieces of filter paper on each side of the sandwich. Make sure all air bubbles are removed since they will affect the efficiency of the electroblotting.
5. Electrotransfer proteins from the gel on the membrane for ~60 min at room temperature. Maintain the current at 0.8 mA per 1 cm² of the gel area and limit the voltage to 15 V.
6. When the transfer is complete, turn off the power and peel off the layers of the sandwich until you reach the membrane. Remove the membrane with forceps and rinse it in deionized water.

Monitoring the Protein Transfer
The efficiency of electrotransfer can be monitored using prestained protein ladders (Spectra™ Multicolor Broad Range Protein Ladder, PageRuler™ Prestained Protein Ladder, PageRuler™ Plus Prestained Protein Ladder and Prestained Protein Molecular Weight Marker. The use of the DualColor™ Protein Loading Pack also allows for monitoring of Western blot protein transfer from gel to membrane. Alternatively, the extent of protein transfer can be determined by staining the polyacrylamide gel after the transfer or by staining the protein directly on the membrane. Proteins on PVDF membranes can be visualized with the PageBlue™ Protein Staining Solution, while Ponceau S, India Ink or Amido Black are recommended for nitrocellulose and PVDF membranes.

Figure. Blotting with a semi-dry transfer unit.

Related products

• ProteoJET™ Mammalian Cell Lysis Reagent
• ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit
• ProteoBlock™ Protease Inhibitor Cocktail
• ProteoJET™ Membrane Protein Extraction Kit
• Bradford Reagent, ready-to-use
• Bovine Gamma Globulin Standard Set, ready-to-use
• Bovine Serum Albumin Standard Set, ready-to-use
• Protein Standard Solution
• DualColor™ Protein Loading Buffer Pack
• Spectra™ Multicolor High Range Protein Ladder
• Spectra™ Multicolor Low Range Protein Ladder
• Spectra™ Multicolor Broad Range Protein Ladder
• PageRuler™ Plus Prestained Protein Ladder
• PageRuler™ Prestained Protein Ladder
• Prestained Protein Molecular Weight Marker
• PageRuler™ Unstained Protein Ladder
• PageRuler™ Unstained Broad Range Protein Ladder
• PageRuler™ Unstained Low Range Protein Ladder
• Unstained Protein Molecular Weight Marker
• PageSilver™ Silver Staining Kit
• PageBlue™ Protein Staining Solution
• 10X Tris-glycine-SDS Electrophoresis Buffer
• 10X Tris-glycine Electrophoresis Buffer
• 10X Tris-tricine-SDS Electrophoresis Buffer
• DTT