What are the most used loading controls for Western Blotting?
Loading control antibodies are some of the most utilised antibodies in research. Antibodies against several different targets are commonly used and it’s not always obvious which you should choose. We provide a guide to some of the most commonly used types of loading control antibodies.
Why do I need a loading control for Western blotting?
- Loading controls are essential for publication worthy Western Blots. They allow you to check that protein loading is the same across the gel, the expression level of the loading control should not vary between the different sample lanes.
- For example, they can be used to check that there has been even transfer from the gel to the membrane across the whole gel.
- This helps to protect against what is known as the “edge effect”, that is when large numbers of lanes are being used, proteins in the outer lanes transfer to the membrane in a position close to the frame. This can result in more intense stains at the edge than in the middle of the gel. Loading controls can show if this has occurred and allows for this variation to be accounted for.
- Once you have established there are not large differences in protein levels, loading controls can then be used to normalise small variations in levels. They can be used to quantify the protein amounts in each lane and normalise the expression levels of your protein of interest to the loading control
What to look for in a loading control?
What makes a good loading control you ask? You should look for proteins that exhibit high level, constitutive expression in the cell type you are examining, as this means it will have constant high expression levels. For this reason, ‘housekeeping’ genes are frequently chosen as loading controls. Secondly, your loading control protein should have a different molecular weight than the protein of interest, this ensure the bands are distinct and expression levels of your protein are quantifiable.
So to help you out, we thought we’d outline our top nine loading controls, and give you links to searches for these loading controls here on CiteAb to ensure you find the best one.
Whole cell and cytoplasmic proteins
- Actin
Suitable for – Whole cell and cytoplasmic extracts
Not suitable for – Skeletal muscle samples
Molecular weight – 43kD
Brief Description – Actin comes in three main different forms. Alpha-actin is a major component on the contractile apparatus in skeletal muscle. Beta and gamma actins are present in most cell types, where they are responsible for cell trafficking, motility and structure.
Actin is found in all eukaryotic cells except for nematode sperm. Their male gametes can’t swim, but rather make amoebic movements to move towards the egg. Unusually, they do not use actin for this, which is the most common protein used to make amoebic movements in cells, they use the MSP protein.
- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Suitable for – Whole cell and cytoplasmic extracts
Molecular weight – 30-40kD
Brief Description – Serves to break down glucose and carbon molecules by acting as a catalyst during the sixth step of glycolysis. Found in most tissue and cell types although expression can differ between tissues.
Hypnoxia, diabetes and some types of cancer can increase GAPDH expression.
- Tubulin
Suitable for – Whole cell and cytoplasmic extracts
Molecular weight – 50 – 55kD
Brief Description – The major component of microtubules, tubulin is highly and consistently expressed across species. Microtubules are involved in maintaining cell shape and positioning of organelles. They are also important in cell division including the formation of mitotic spindles.
Antimicrobial and anti-mitotic drugs can affect tubulin expression.
Mitochondrial proteins
- Voltage-dependent anion-selective channel protein 1 (VDAC1)/Porin
Suitable for – Mitochondrial proteins
Molecular weight – 31kD
Brief Description – Voltage-dependent anion-selective channel protein 1 is also named porin 31 HL/HM or plasmalemmal porin. Expressed throughout tissues and conserved across species, VDAC1 are beta barrel proteins that cross the cellular membrane, acting as a pore that molecules can diffuse through.
- Cytochrome C Oxidase (COX IV)
Suitable for – Mitochondrial proteins
Molecular weight – 16kD
Brief Description – The COX IV (said “Cox 4”) antibody is reactive with the COX IV protein, generally expressed at a consistent high level, this antibody makes an effective loading control for mitochondria. However, be aware that many proteins run at the same molecular weight as COX IV, VDAC1 makes a good alternative mitochondrial loading control for proteins of this size.
Nuclear envelope proteins
- Lamin B1
Suitable for – Nuclear envelope proteins
Molecular weight – 66kD
Brief Description – The lamin family of proteins make up the nuclear lamina- a two-dimensional matrix of proteins found next to the inner nuclear membrane. Highly conserved across species, lamin proteins are thought to be involved in nuclear stability, chromatin structure and gene expression. This loading control is not suitable for experiments where the nuclear envelope has been removed.
- TATA binding protein (TBP)
Suitable for – Nuclear proteins
Molecular weight – 38kD
Brief Description – The TATA binding protein, (TBP) is a transcription factor that binds specifically to the TATA box, a DNA sequence found in the promoter region of archaeal and eukaryotic genes. It helps position RNA polymerase II over the transcription site at the start of the gene, but is only found in 10-20% of human promoters that have TATA boxes, so is probably not the only protein involved in RNA polymerase II positioning.
- Histone H2B
Suitable for – Nuclear proteins
Molecular weight – 14kD
Brief Description – A basic histone involved in the nucleosome structure of chromatin in eukaryotes, Histone H2B has a main globular domain and a long N terminal tail.
Search for Histone H2B on CiteAb
- Proliferating Cell Nuclear Antigen (PCNA)
Suitable for – Nuclear proteins
Molecular weight – 36kD
Brief Description – A highly conserved protein, PCNA helps to increase processivity of leading strand synthesis during DNA replication. PCNA is quickly degraded when DNA damage pathways are activated.
So, that’s all from us – please leave a comment if there are any loading controls you would highly recommend.
– Eleanor and the CiteAb team
References
http://www.actrec.gov.in/histome/histones.php?histone=H2B
http://protocolsonline.com/protein-science/electrophoresis/loading-controls/
http://www.antibodies-online.com/resources/17/1217/Loading+controls/
http://www.novusbio.com/research-areas/cellular-markers/loading-controls.html
http://www.abcam.com/cox-iv-antibody-mitochondrial-loading-control-ab16056.html
http://blog.ptglab.com/index.php/western-blot-loading-controls/
