Loading Control Antibodies

Loading Control Antibodies

What is a Loading Control?

A loading control is a protein which is ubiquitously expressed in the cell. Its concentration rarely varies and it is often a protein expressed abundantly in a particular cell type. Loading controls are used as an internal standard in western blotting experiments. Western blot works on the premise that a protein/proteins in a sample are electrophoretically separated on a gel based on their size. These are then transferred to a solid surface, such as a nitrocellulose membrane, and treated with primary and secondary antibodies which allow for the determination of concentration of the target protein. Loading controls are used to calculate the concentration of a protein/proteins in a sample. It is the use of loading control antibodies that allows for the normalization of results as target protein concentration is compared relatively to loading control protein concentration.


Why are Loading Controls used?

  • Experimental failures that may be introduced via human error (uneven quantity of protein added to well) can be almost immediately distinguished. This improves efficiency and reduces time of experiments as there is no time wasted dealing with inaccurate or inconclusive results.

  • Loading controls are used to establish if there has been even transfer not only of the sample, but also of the gel across the membrane. This is another common issue associated with western blot.

  • The use of a loading control can also correct for the ‘edge effect’. This is when proteins added to wells at the sides of the gel drift in towards the centre. Loading controls can establish and control for this.

Figure 1. Immunofluorescence analysis of HeLa cells using ACTB antibody (CABC006) at dilution of 1:100 (40x lens). Blue: DAPI for nuclear staining.


Types of Loading Controls

Popular loading controls used may be housekeeping gene proteins or other proteins that are expressed constitutively across all cell types. Loading controls are proteins that are highly conserved, highly expressed, and stabile under most experimental conditions. Expression is rarely affected by biological or experimental variations.

Loading Control Molecular Weight Uses Limitations

Beta Actin

43 kDA

Whole cell and cytoplasmic extracts

May exhibit variations in expression in some cell/tissue types. Not suitable as a loading control in tissues such as skeletal muscle where its expression is very high. Not suitable for studies involving subjects with a large age difference. Cannot be used as loading control of nuclear extracts as Beta actin is a component of chromatin remodeling complexes.

Glyceraldehyde 3-phosphatase
dehydrogenase (GAPDH)

35 kDA

Whole cell and cytoplasmic extracts

Not suitable for oxygen-related studies (hypoxia causes an upregulation of GAPDH. May exhibit variations in expression in some cell/tissue types or disease states.

Alpha Tubulin

50 kDA

Whole cell and cytoplasmic extracts

Expression may be affected by anti-fungal or anti-cancer drugs. Not suitable for studies involving subjects with a large age difference.

Beta Tubulin

50 kDA

Whole cell and cytoplasmic extracts

Expression may be affected by anti-fungal or anti-cancer drugs.Not suitable for studies involving subjects with a large age difference.

TATA-binding protein (TBP)

38 kDA

Nuclear extracts

Not suitable as a loading control if nuclear components of cell have been removed from sample.

Cyclophilin A

19 kDA

Nuclear extracts

Not suitable as a loading control if nuclear components of cell have been removed from sample.

Immunoglobulin G (IgG)

50 kDA - Heavy chain

25 kDA - Light chain

Serum extracts. Used to control for non-specific protein binding to Fc receptors of antibodies during western blot analysis.

Not suitable as a loading control of cytosolic/nuclear extract samples.


Limitations of Loading Controls

Although under most conditions loading control proteins do not vary in expression there may be some exceptions. These include:

  • Loading control proteins may vary in expression across different tissue types (higher beta actin expression in skeletal muscle vs. elsewhere in the body).
  • Loading controls proteins may vary in expression in different disease states (e.g. cancer).
  • Loading controls proteins may vary in expression depending on age of subject from which the sample was taken.
  • The loading control of choice must have a different molecular weight than the protein of interest so that the researcher can distinguish between the bands when performing western blot.

What are Loading Control Antibodies?

Loading control antibodies are antibodies with reactivity against the loading control protein of interest. They must be highly specific to be effective in the role of quantifying housekeeping gene proteins. In this way they consequently allow for the relative quantification of the protein(s) of interest within the sample.

Figure 2. Western blot analysis of extracts of various cell lines, using HRP-Conjugated-Beta-Tubulin antibody (CABC030) at 1:10000 dilution.Secondary antibody: HRP Goat Anti-Mouse IgG (H+L) (CABS003) at 1:10000 dilution.Lysates/proteins: 25ug per lane.Blocking buffer: 3% nonfat dry milk in TBST.Detection: ECL Basic Kit (AbGn00020).Exposure time: 1s.


Antibody Genie Loading Control Antibodies

SKU Product Name

ACTB Monoclonal Antibody

ACTB Polyclonal Antibody

alpha Tubulin Monoclonal Antibody (TUBA4A)

alpha Tubulin Monoclonal Antibody (TUBA1B) (Human, Mouse, Rat)

alpha Tubulin Polyclonal Antibody (TUBA1B) (Mouse)

alpha Tubulin Polyclonal Antibody (TUBA1B) (Human, Mouse)

Beta Actin Monoclonal Antibody (IF)

Beta-Tubulin Monoclonal Antibody (IHC)

Beta-Tubulin Monoclonal Antibody (WB, IF)

Beta-Tubulin Monoclonal Antibody (WB, IF, IHC, IP)

Beta-Tubulin Polyclonal Antibody (WB, IHC)

Beta-Tubulin Polyclonal Antibody (WB, IHC, IF)

HRP-conjugated ACTB Monoclonal Antibody

HRP-conjugated Beta-Tubulin Monoclonal Antibody

Mouse Control IgG

Plant actin Monoclonal Antibody

Rabbit Control IgG

Rat Control IgG

TUBA4A Polyclonal Antibody (WB)

TUBA4A Polyclonal Antibody (WB, IHC, IF)

TUBA4A Polyclonal Antibody (WB, IHC)

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