![]() ![]() CD133 is a highly glycosylated protein with an apparent molecular weight of 115-120 kDa. One commonly used enzyme is PNGase F it removes N-linked glycans by cleaving the bond between the innermost N-Acetylglucosamine of the glycan chain and the asparagine residue.ĬD133, also known as PROM1 (prominin-1) (18470-1-AP), is a transmembrane glycoprotein with an NH2-terminal extracellular domain, five transmembrane loops, and a cytoplasmic tail. WB protein species from the digested sample are then compared with the undigested sample, and any observed shift in molecular weight indicates protein glycosylation. Prior to WB, the protein sample is incubated with an enzyme that is able to remove parts or full glycan chains. Please note: An enzymatic deglycosylation is a commonly used experimental technique to verify whether a studied protein is glycosylated. The signal peptide is cleaved off during protein transport to the plasma membrane and the protein is heavily N-glycosylated with an apparent molecular weight of 45–70 kDa with the major glycosylated form of 45–50 kDa (PMID: 27572267). Extensive glycosylation adds additional molecular weight, not included in the original protein sequence, which makes proteins migrate slower.įigure 3: Programmed cell death ligand 1 (PD-L1, CD274, or B7-H1) (66248-1-Ig) is a type I transmembrane protein, acting as a key regulator of the adaptive immune response. The two most common types of glycosylation in Eukaryotes are N-linked glycosylation – to asparagine, and O-linked glycosylation – to serine and threonine. That means a covalent attachment of sugar moieties is added to the polypeptide chain. The majority of proteins that are synthetized on ribosomes associated with the endoplasmic reticulum undergo glycosylation. Posttranslational modifications (PTMs) a) Glycosylation and glycanation Besides, MMP9 can also exist as a dimer of 180 kDa (PMID: 7492685). It gets sequentially cleaved by MMP3 into a processed form of 68 kDa through an intermediate form – 78/82 kDa (PMID: 1371271). Most MMPs are secreted as inactive proproteins that are activated when cleaved by extracellular proteinases. Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of the extracellular matrix in many physiological processes, including embryonic development, reproduction, tissue remodeling, and disease processes such as arthritis or metastasis. Caspase 3 (19677-1-AP) exists as an inactive proenzyme form of 32 kDa (p32), which upon apoptotic signaling gets cleaved into two active subunits (p19/17 and p12) that assemble into a functional tetrameric enzyme (PMID: 7596430). PINK1 is further transferred into the inner membrane where it is cleaved into a 52 kDa mature form.Ĭaspases, a family of endoproteases, are critical players in cell regulatory networks controlling inflammation and cell death. The precursor of PINK1 (65 kDa) is synthesized in the cytosol and is imported into the outer membrane of mitochondria. Protein precursors need to be processed by proteases in order to engender a functional product (without pro-peptide).įigure 2: PINK1 (23274-1-AP) is a mitochondrial serine/threonine-protein kinase that protects cells from stress-induced mitochondrial dysfunction. Additionally, a subset of proteins has pro-peptides – protein domains that are present in protein precursors. They are usually well annotated in protein databases, e.g., UniProt. The presence of signal peptides can be predicted by various online tools or based on previously published data. This results in the mature protein running at a lower than predicted molecular weight. They are often cleaved by various proteases during their subcellular transport. length located predominantly at their N-termini. Many proteins that undergo transport through the secretory pathway have signal peptides of 15–35 aa. Signal peptide (and a pro-peptide) gets cleaved off Read below for more detail on each possible cause.Ĭommon causes of unexpected size of western blot band 1. Here we have summarized the most common reasons for why this may occur (Figure 1). Often the calculated MW is different from that observed on the WB. It is easy to calculate, e.g., using the free online ExPASy tool. The predicted molecular weight (MW) of the protein is the sum of the molecular weights of all the protein's amino acids. This means smaller proteins migrate faster than larger ones. Because the SDS-PAGE is conducted in denaturing conditions, proteins migrate according to their molecular weights irrespective of their secondary/tertiary structure, charge or protein–protein interactions. The first step in western blotting is sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer on a membrane and subsequent detection with specific antibodies. Signal peptide (and a pro-peptide) gets cleaved offĢ. ![]()
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