Western blotting is a commonly used method to determine specific protein levels in a sample (e.g. tissue homogenate, extract). Loading control antibodies are important controls as they indicate the equal loading of samples across all wells. Loading controls also indicate the proper transfer of proteins to the membrane during the western blotting process. Loading controls are typically proteins with high and ubiquitous expression. The expression levels of the loading controls should be consistent regardless of tissue or cell type or experimental conditions.
Loading Controls - normalizing protein levels in western blots
|Suitability||Name||Molecular Weight (kDa)|
|Whole cell & cytoplasmic||Actin||~ 42|
|Whole cell & cytoplasmic||GAPDH||~ 37|
|Whole cell & cytoplasmic||Tubulin||50 - 55|
|Mitochondrial||VDCA1 / Porin||31|
Suitability: Whole cell & cytoplasmic extracts
Molecular weight: ~42 kDa
Actins are one of the most highly-conserved proteins. Actins are comprised of three main isoform groups: the alpha, beta and gamma. The alpha actins are found in muscle tissues and are a major component of the contractile apparatus. The beta and gamma actins are present in most cell types as parts of the cytoskeleton and are mediators of cell trafficking, structural integrity and cell motility.
Note: Changes in cell growth conditions and interactions with extracellular matrix components have been found to alter actin protein synthesis.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Suitability: Whole cell & cytoplasmic extracts
Molecular weight: ~37 kDa
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serves to break down glucose for energy and carbon molecules by catalyzing the sixth step of glycolysis. It is also involved in transcription, RNA binding and transport, DNA replication and repair, as well as apoptosis and ER to Golgi vesicle shuttling. GAPDH is highly and constantly in most tissues and cell types although its expression can differ between tissues.
Note: Hypoxia, diabetes and some types of cancers can increase GAPDH expression in certain cell types.
Molecular weight: 14 kDa
Histone H2B is one of the five basic histones (H1, H2A, H2B, H3 and H4) involved in the structure of chromatin in eukaryotic cells. Histone H2B has a main globular domain and a long N terminal tail which is a part of the structure of the ?beads on a string? nucleosome structure. Histones are present in abundance in virtually all eukaryotic cells.
Note: Histone doubles prior to cell division and therefore not suited for comparing S phase with with pre-S phase cells.
Histone H2B antibodies
Proliferating Cell Nuclear Antigen (PCNA)
Molecular weight: 36 kDa
Proliferating Cell Nuclear Antigen (PCNA) is a highly conserved protein. PCNA is a cofactor of DNA polymerase ? in eukaryotic cells and helps to increase the processivity of leading strand synthesis during DNA replication. PCNA is an example of a DNA clamp as it encircles the DNA, (which, as a result, creates topological link to the genome).
Note: PCNA is quickly degraded when DNA damage pathways are activated.
TATA binding protein (TBP)
Molecular weight: 38 kDa
The TATA binding protein (TBP) is widely expressed, though its levels are highest in the testis and ovary. TBP is a transcription factor that binds specifically to the TATA box. The TATA box is a DNA sequence found in the promoter region of archaeal and eukaryotic genes. TBP is a component of the TFIID, a DNA-binding multiprotein transcription factor that in turn makes up part of the RNA polymerase II preinitiation complex (PIC).
Note: TBP is not a suitable loading control when DNA and other nuclear components have been removed.
Suitability: Whole cell/cytoplasmic
Molecular weight: 50 - 55 kDa
Tubulins are highly and stably expressed and conserved across species. Tubulins are the major components of microtubules. Microtubules are comprised of dimers of alpha and beta tubulin. Microtubules are involved in maintaining the cell shape as well as various cell processes such as positioning of cell organelles, intracellular transport and extracellular transport by flagella and cilia. They are also involved in cell division (mitosis and meiosis) including the formation of mitotic spindles.
Note: Tubulin expression may vary according to resistance to antimicrobial and antimitotic drugs.
Voltage-dependent anion-selective channel protein 1 (VDCA1/Porin)
Molecular weight: 31 kDa
Voltage-dependent anion-selective channel protein 1 (VDAC1), also named porin 31HM, porin 31HL or plasmalemmal porin, is universally expressed throughout tissues and conserved across species. It is found in the outer membrane of Gram-negative bacteria, the mitochondria as well as the chloroplast. VDCA1/Porin are beta barrel proteins that cross a cellular membrane and act as a pore that molecules can diffuse through.
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