Loading control antibodies are helpful for assessing western blotting efficiencies and comparing the amounts of protein loaded in each well across a gel.

These controls help determine whether sample-to-sample expression level differences are due to actual protein levels in a given cell lysate or from loading variances. The best loading controls are proteins with high level, constitutive expression in the cell.

Loading controls also allow you to assess whether the transfer from the gel occurred evenly across the whole western blotting membrane.

Loading control antibodies can also be used as complementary antibody stains in immunofluorescence studies with proteins of interest. It is essential to have reliable loading controls for a high impact journal publication.

To help you choose the right loading control antibodies, below is a list of the most commonly used ones along with some sample stainings.

Beta actin antibodies

Beta actin antibodies provide a specific and useful tool for studying the intracellular distribution of beta actin and the static and dynamic aspects of the cytoskeleton.

Beta actin (42 kDa) is ubiquitously expressed in all eukaryotic cells and is therefore frequently used as a loading control.

Beta actin is one of six isoforms of actin that have been identified in mammals.

Actin is the primary component of the cytoskeleton, and beta actin is predominantly expressed in non-muscle cells, where it controls cell structure and motility.

beta actin 1
Immunohistochemical analysis of beta actin showing staining in the cytoskeleton of paraffin-embedded human breast carcinoma (right), compared with a negative control without primary antibody (left). The staining of the beta actin with the loading control antibody (Cat. No. MA5-15739-BTIN) confirms that proteins in the sample are accessible for binding by other primary antibodies.

beta actin 2
Western blot analysis of beta actin was performed by loading 30 μg of various cell lysates in wells of a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and probed with an HRP-conjugated beta actin monoclonal antibody (Cat. No. MA5-15739-HRP). Chemiluminescent detection was performed using SuperSignal™ West Pico Substrate (Cat. No. 34080).

Find out more about Quality Invitrogen™ beta actin antibodies on Thermo Fisher website. These antibodies are also offered as fluorescent and enzyme-conjugated versions.

Beta tubulin antibodies

Beta tubulin is a 55 kDa protein that forms a heterodimer with alpha tubulin to make tubulin, the primary component of microtubules.

Microtubules mediate many cell movements, including intracellular transport, cilliary and flagellar beating, chromosome alignment in mitosis and meiosis, and structural support of the cytoskeleton.

Beta tubulin is ubiquitously expressed in eukaryotic cells and is often used as a loading control.

beta tubulin 1
Immunohistochemical analysis of formalin-fixed, paraffin-embedded human kidney tubules stained using a beta tubulin polyclonal antibody (Cat. No. PA1-41331).

beta tubulin 2
Western blot analysis of beta tubulin was performed by loading various cell lysates onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a low fluorescence PVDF membrane and probed with a beta tubulin monoclonal antibody conjugated to Alexa Fluor™ 555 dye (Cat. No. MA5-16308-A555).

Find out more about Quality Invitrogen™ beta tubulin antibodies on Thermo Fisher website. These antibodies are also offered as fluorescent and enzyme-conjugated versions.

Cyclophilin D

Cyclophilin D (CyPD) is a member of the cyclophilin family of peptidylprolyl isomerases.

Cyclophilin D (40 kDa) is located in the matrix of the mitochondria where it acts as an integral member of the permeability transition pore complex, which also includes the voltage dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). Activation of this pore is involved in the induction of apoptotic and necrotic cell death.

Cyclophilin D is conserved, ubiquitous, and abundantly expressed in most cells, making it a good loading control.

cyclophilin d 1
Immunohistochemical analysis was performed on deparaffinized human hepatocarcinoma biopsy tissues. Tissues were probed with a rabbit polyclonal antibody recognizing cyclophilin D (Cat. No. PA3-022) or treated equivalently without primary antibody (negative control). Tissues were counterstained with hematoxylin and prepared for mounting.

cyclophilin d 2
Western blot analysis was performed on whole cell extracts. The blots were probed with Anti-Cyclophilin D Rabbit Polyclonal Antibody (Cat. No. PA3-023) and detected by chemiluminescence using Goat Anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Cat. No. G-21234). A 40 kDa band corresponding to cyclophilin D was observed across the cell lines tested; additional bands at 21 and 35 kDa may correspond to other cyclophilin isoforms.

 

Find out more about Quality Invitrogen™ cyclophilin D antibodies on Thermo Fisher website.

Glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its role in glycolysis.

GAPDH catalyzes the phosphorylation of glyceraldehyde-3-phosphate. In addition to this metabolic function, GAPDH is involved in many nonmetabolic cellular processes, including membrane fusion, transcription, RNA transport, DNA replication, and apoptosis.

GAPDH has been associated with various cancers and may also play a part in neurodegenerative diseases like Alzheimer’s and Huntington’s diseases. While it is expressed differentially from tissue to tissue, GAPDH is often stably and constitutively expressed, making it useful as a loading control.

gapdh 1
Immunohistochemical analysis of GAPDH showing staining in the nucleus and cytoplasm of paraffin-embedded human kidney tissue (right), compared with a negative control without primary antibody (left). The cells were probed with a GAPDH monoclonal antibody (Cat. No. MA5-15738).

gapdh 2
Western blot analysis of GAPDH was performed by loading 75 µg of various cell lysates per well. The membrane was probed with a GAPDH monoclonal antibody conjugated to DyLight™ 680 dye (Cat. No. MA5-15738-D680).

Find out more about Quality Invitrogen™ GAPDH antibodies on Thermo Fisher website. These antibodies are also offered as fluorescent and enzyme-conjugated versions.

Loading control antibodies are available in unconjugated form, or conjugated to biotin, HRP, DyLight™ dyes, or Alexa Fluor™ dyes.

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