Protein electrophoresis and western blot
Protein electrophoresis and western blot are two consecutive steps of the same workflow: the run separates the proteins of an extract by molecular weight or isoelectric point, the blot transfers them to a membrane and makes them accessible to an antibody. The reagents needed to walk through it — lysis buffers, quantification systems, separation matrices, stains, markers, membranes and blockers — come in the CABRU catalogue from G Biosciences and FineTest: G Biosciences is present with 6,604 references in total, 210 of them tied to electrophoresis and blotting and 190 organised as kits.
The points where the outcome is decided
Almost every western blot failure comes from choices made before the membrane. The lysis buffer determines what is extracted and constrains the quantification method; the gel percentage decides whether the expected band falls in the resolved zone; membrane and blocker decide how much background reaches the detection step. These decisions are independent of the antibody, and they are worth making before an antibody is called into question.
| Decision point | Options | How to decide |
|---|---|---|
| Protein quantification | Bradford, BCA | Bradford does not tolerate detergents; BCA is affected by reducing agents and chelators. The lysis buffer decides |
| Gel percentage | 7.5% to 15%, or gradient | High percentages resolve small proteins, low ones large proteins; a gradient covers a wide range |
| Membrane | Nitrocellulose, PVDF | PVDF must be pre-wetted in methanol and binds more protein; nitrocellulose is ready to use but more fragile |
| Blocking agent | Skimmed milk, BSA, protein-free blockers | Milk contains phosphorylated casein: it is not used with anti-phospho antibodies |
| Molecular weight marker | Prestained, unstained | Prestained can be followed during run and transfer; unstained gives the more accurate weight estimate |
1D and 2D: when the second dimension is needed
One-dimensional SDS-PAGE separates by mass alone: proteins of similar weight but different charge end up in the same band. The two-dimensional version puts isoelectric focusing first, so the first dimension separates by isoelectric point and the second by mass; this is how isoforms and proteins carrying charge-shifting post-translational modifications, such as phosphorylated forms, are told apart. The cost is a longer protocol, more sensitive to sample quality and to salts, ionic detergents and nucleic acids, which have to be removed before focusing.
What the catalogue holds
In the G Biosciences range, 120 references cover blotting and membranes, 114 agarose matrices for electrophoresis, 73 stains — 8 of them Coomassie-based — 53 extraction and lysis reagents, 44 protein quantification systems, 12 acrylamide formulations and 6 ladders and markers, one of them a prestained protein molecular weight marker. The full list, with codes and pack sizes, can be browsed in the product catalogue filtering by company. When a reference is not on the list, its existence is checked with the manufacturer.