Loading Controls

loading control

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Western Blot Protocol

Western Blot Troubleshooting Guide

Western Blot Video Protocol

HRP Conjugated Secondary Antibodies

Western Blot Membranes

Western Blot Lysates

Loading Controls

What is a loading control?

  • Loading Control: A loading control is an antibody specific for a highly or ubiquitously expressed protein and can be used to normalize protein levels in western blot.

Loading Control Cell

What should I consider when choosing a loading control for my experiment?

  • Detection Size: Choosing a loading control that is close in size, but still large or small enough to be easily distinguished from your target protein is crucial. If the target protein and loading control are the same size, visualization will be impaired and data interpretation compromised.

  • Strong Expression: Ensure the chosen loading control demonstrates strong expression in your sample. Most common loading control targets are highly expressed constitutive genes required for basic cellular processes and cellular vitality (housekeeping genes). However, the expression of some rarer controls may not be as ubiquitous.

  • Expression Factors: Choose a loading control whose expression is not affected by an experimental variable (e.g. if studying a metabolic process use of GAPDH as a loading control is not recommended due its role in glycolysis). An optimal loading control should detect a globally expressed protein that is not differentially regulated in normal and disease states.

When do I need to use a loading control antibody?

  • Quantitative Comparisons: Loading controls are necessary when comparing signal between wells. Observed changes in target protein expression can only be considered true if expression of the loading control is equal across wells.

  • Loading and Transfer Confirmation: Loading controls confirm all samples have been loaded and transferred equally across wells. Uneven transfer from the gel to membrane can be determined by analysis of loading control signal.

  • Publishing your study: Most journals require loading control data in western blot analysis to ensure differential expression between experimental samples is real. Loading controls normalize target protein expression across all wells.


Common Loading Controls

Subcellular Localization

Loading Control

Molecular Weight (kDa)

Cytoplasm/Whole Cell

Alpha-tubulin

Beta-actin

GAPDH

Cyclophilin B

55

43

37

21

Mitochondria

HSP60

COX IV

60

17

Nucleus

Lamin B1

HDAC

PCNA

Histone H3

66

60

29

17

Serum Transferrin 77

Loading Control Tips

Target Protein

Note

Beta-actin Not suitable for nuclear extract as beta-actin is a component of chromatin remodeling complexes (1). May not be suitable for studies involving subjects with a large age difference (2).
GAPDH Not suitable for oxygen-related studies as hypoxia can upregulate GAPDH expression (3). May not be suitable for studies involving subjects with a large age difference (2).
Alpha-tubulin May not be suitable for studies involving subjects with a large age difference (2). Tubulin expression can be affected by anti-cancer and anti-fungal drugs.
Lamin B1 Not suitable for embryonic stem cells (4).
PCNA Not suitable for serving as a loading control for non-proliferating cells.
COX IV Many proteins run at around 15~17 kDa; hence, it may be necessary to consider an alternative control antibody if your protein of interest is similar in size to COX 4.
Transferrin Transferrin levels can be influenced by some disease states and treatments such as retinoic acid.
Histone H3 Many proteins run at around 15~17 kDa; hence, it may be necessary to consider an alternative control antibody if your protein of interest is similar in size to Histone H3.

View All Loading Control Antibodies

PMID:

  1. 12045110
  2. 25429098
  3. 10364451
  4. 22116031