Tag: 2D-gel Two-Dimensional Polyacrylamide Gel Electrophoresis

2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis) is an advanced technique used to separate complex mixtures of proteins with very high resolution. It separates proteins based on two independent properties:

| 1st Dimension | Isoelectric Point (pI) |
| 2nd Dimension | Molecular Weight (kDa) |

🔬 How 2D-PAGE Works

2D-PAGE involves two sequential electrophoresis steps:

STEP 1 – Isoelectric Focusing (IEF)

• Proteins are applied to a strip containing a pH gradient (Immobilized pH gradient strip — IPG).

• When voltage is applied, proteins migrate until they reach the pH where their net charge becomes zero (isoelectric point)

• Result: Proteins are separated horizontally according to pI differences.

STEP 2 – SDS-PAGE

• The focused strip from Step 1 is placed on top of an SDS polyacrylamide gel.

• SDS denatures proteins and gives them a uniform negative charge relative to size

• Proteins migrate vertically based on molecular weight

• Result: Proteins are separated vertically according to size.

🧠 Final Output & Interpretation

The final gel shows protein spots instead of bands.

Each spot = a distinct protein

• X-axis: pI difference (left to right)

• Y-axis: molecular weight (top to bottom)

Protein Identification

After separation, individual spots can be:

• Cut out from the gel

• Digested with trypsin

• Analyzed by Mass spectrometry (MALDI-TOF / LC-MS/MS)

📌 Applications of 2D-PAGE