Antibodies to Loading Controls - FocusOn 117

Acris Antibodies offers a wide range of antibodies to proteins which are suitable for use as loading controls. The ‘Product list’ shows a selection of our recommended antibodies.

Introduction

Western blot analysis can be used for (semi-) quantitative detection of target proteins, especially concerning experimental procedures which lead to altered expression levels of the target protein.
To compare the effect of different treatments on the target protein a method to standardize the amount of total protein loaded per lane and to confirm that equal amounts were loaded in each lane of the gel is needed.
For this purpose so called ‘house keeping’ proteins are selected as controls. The loading control ideally should not show an altered expression level during the course of the experiment so one has to carefully choose the appropriate loading control for each experimental setup. Proteins selected as loading controls are often of fundamental importance in maintaining basic cellular functions.

Actin beta

In vertebrates 3 main groups of actin isoforms, alpha, beta and gamma, have been identified. The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus. The beta and gamma actins coexist in most cell types as components of the cytoskeleton and as mediators of internal cell motility. Beta actin (ACTB) is a highly conserved protein that is involved in cell motility, structure and integrity. Beta actin is a cytoplasmic protein ubiquitously expressed in all eukaryotic cells and therefore often used as a loading control.

Fig. 1: Western blot analysis of beta actin in decreasing amounts of human ovary tissue lysate using Cat.-No. AP21589PU-N: A) 40 µg, B) 30 µg, C) 20 µg, and D) 10 µg per lane
Fig. 1: Western blot analysis of beta actin in decreasing amounts of human ovary tissue lysate using Cat.-No. AP21589PU-N: A) 40 µg, B) 30 µg, C) 20 µg, and D) 10 µg per lane

Tubulin alpha and beta

Heterodimers consisting of alpha- and beta-tubulin form microtubules of the eukaryotic cytoskeleton. Tubulin beta5 (TUBB5) and tubulin alpha (1B chain/TUBA1B) are ubiquitously expressed representing a good choice as loading controls for cells of multiple origins.
Class III beta tubulin (TUBB3/TUBB4) is a vertebrate tubulin isotype only found in neurons. Exception to the neuro-specificity of beta III tubulin occurs in cells in the mammalian testis during cancer development. Therefore, TUBB3 can be used as a loading control for neuronal samples.
Tubulins are generally highly conserved proteins throughout all eukaryote species. Most available antibodies to tubulins are reactive on nearly all species tested so far.

Fig. 2: Western blot using affinity purified alpha-tubulin antibody Cat.-No. AP09289PU-N shows detection of a predominant band at ~50 kDa corresponding to alpha tubulin (arrowhead). Lanes correspond to whole cell lysates from mouse brain (lane 1), rat brain (lane 2), A431 cells (lane 3), Jurkat cells (lane 4) and HeLa cells (lane 5)
Fig. 2: Western blot using affinity purified alpha-tubulin antibody Cat.-No. AP09289PU-N shows detection of a predominant band at ~50 kDa corresponding to alpha tubulin (arrowhead). Lanes correspond to whole cell lysates from mouse brain (lane 1), rat brain (lane 2), A431 cells (lane 3), Jurkat cells (lane 4) and HeLa cells (lane 5)
Fig. 3: Western blot analysis of beta tubulin (arrow-head) in A: human brain, B: mouse brain and C: rat brain tissue lysates using Cat.-No. AP21644PU-N
Fig. 3: Western blot analysis of beta tubulin (arrow-head) in A: human brain, B: mouse brain and C: rat brain tissue lysates using Cat.-No. AP21644PU-N

GAPDH

Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) is well known as one of the key enzymes involved in glycolysis. GAPDH, which is constitutively expressed in almost all tissues at high levels, is suitable as an excellent loading control.

Staining of Rh30 rhabdomyosarcoma (upper panels) and SJSA-1 Ewing’s sarcoma cells (lower panels) for GAPDH with ACR001P anti-GAPDH monoclonal antibody (left panels). FITC conjugated secondary antibody. The middle panels show DAPI staining of the cell nuclei. The right panels show merged images.
Fig. 4: Staining of Rh30 rhabdomyosarcoma (upper panels) and SJSA-1 Ewing’s sarcoma cells (lower panels) for GAPDH with ACR001P anti-GAPDH monoclonal antibody (left panels). FITC conjugated secondary antibody. The middle panels show DAPI staining of the cell nuclei. The right panels show merged images.
Western Blotting for GAPDH on Rat Brain lysates using anti-GAPDH monoclonal antibody Cat.-No ACR001P at 1 µg/ml (Top) and 0.1 µg/ml (Bottom).
Fig. 5: Western Blotting for GAPDH on Rat Brain lysates using anti-GAPDH monoclonal antibody Cat.-No ACR001P at 1 µg/ml (Top) and 0.1 µg/ml (Bottom).
Western Blotting for GAPDH on HUVEC cell lysate using anti-GAPDH monoclonal antibody Cat.-No ACR001P at 2 µg/ml (A and B) and 0.5 µg/ml (C and D).
Fig. 6: Western Blotting for GAPDH on HUVEC cell lysate using anti-GAPDH monoclonal antibody Cat.-No ACR001P at 2 µg/ml (A and B) and 0.5 µg/ml (C and D).

VDAC

Voltage-dependent anion-selective channel protein 1 (VDAC) is an outer membrane mitochondrial protein. The VDAC proteins are thought to form aqueous channels, or pores, through which adenine nucleotides cross the outer mitochondrial membrane. VDACs have been implicated in the formation of the mitochondrial permeability transition pore complex in apoptotic cells. VDAC is highly expressed in heart, liver and skeletal muscle, where concentrations of mitochondria are at their highest. VDAC can be used as a loading control with mitochondrial preparations as well as whole cell lysates.

Fig. 6: Western blot analysis of VDAC/porin Cat.-No. AP00265PU-N with 3T3 cell lysate
Fig. 7: Western blot analysis of VDAC/porin Cat.-No. AP00265PU-N with 3T3 cell lysate

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Primary Antibodies

Catalog No. Host Iso. Clone Pres. React. Applications  

Actin beta / ACTB (359-368) (Loading Control) antibody

Immunohistochemical staining.Affinity Purified anti-beta Actin antibody at 1:200 is shown to detect actin at the neuromuscular junction of rana pipiens tissue (sections 4.2 µm thick). Rabbit IgG Aff - Purified Hu E, P, WB
0.2 mg / €390.00
  Acris Antibodies GmbH

alpha Tubulin / TUBA1B (Loading Control) antibody

Figure 1. Immunofluorescence staining of 3T3 mouse embryonal fibroblast cell line using anti-alpha-Tubulin antibody (TU-01; green) and anti-Vimentin antibody (VI-01; SM3128P; red). Nucleus is stained with DAPI (blue). Mouse IgG1 TU-01 Purified All Species C, E, ICC/IF, IP, P, WB
0.1 mg / €240.00
  Acris Antibodies GmbH

alpha Tubulin / TUBA1B (Loading Control) antibody

alpha Tubulin / TUBA1B Mouse IgG1 TU-01 Biotin All Species WB
0.1 mg / €310.00
  Acris Antibodies GmbH

alpha Tubulin / TUBA1B antibody

Immunoprecipitation of alpha-tubulin from HeLa and RAJI cell lysate by antibody TU-16 and its detection by antibody TU-01. IgM heavy chain (76-92 kDa) and IgM light chain (25-30 kDa) indicated. Mr of alpha tubulin is around 50 kDa. L = lysate IPr = immunoprecipitate (reducing conditions) IPn = immunoprecipitate (non-reducing conditions) Mouse IgM TU-16 Purified All Species C, E, ICC/IF, IP, P, WB
0.1 mg / €240.00
  Acris Antibodies GmbH

GAPDH (Loading Control) antibody

Staining of Rh30 rhabdomyosarcoma (upper panels) and SJSA-1 Ewing’s sarcoma  cells (lower panels) for GAPDH with ACR001P anti-GAPDH monoclonal antibody (left panels).  FITC conjugated secondary antibody. The middle panels show DAPI staining of the cell nuclei. The right panels show merged images. Mouse IgG1 6C5 Purified Can, Fe, Fi, Hu, Ms, Por, Rb, Rt E, ICC/IF, IP, WB
0.2 mg / €270.00
  Acris Antibodies GmbH

GAPDH (Loading Control) antibody

Staining of Rh30 rhabdomyosarcoma (upper panels) and SJSA-1 Ewing’s sarcoma  cells (lower panels) for GAPDH with ACR001P anti-GAPDH monoclonal antibody (left panels).  FITC conjugated secondary antibody. The middle panels show DAPI staining of the cell nuclei. The right panels show merged images. Mouse IgG1 6C5 Purified Can, Fe, Fi, Hu, Ms, Por, Rb, Rt E, ICC/IF, IP, WB
0.1 mg / €170.00
  Acris Antibodies GmbH

GAPDH (Loading Control) antibody

Staining of Rh30 rhabdomyosarcoma (upper panels) and SJSA-1 Ewing’s sarcoma  cells (lower panels) for GAPDH with ACR001P anti-GAPDH monoclonal antibody (left panels).  FITC conjugated secondary antibody. The middle panels show DAPI staining of the cell nuclei. The right panels show merged images. Mouse IgG1 6C5 Purified Can, Fe, Fi, Hu, Ms, Por, Rb, Rt E, ICC/IF, IP, WB
20 µg / €60.00
  Acris Antibodies GmbH

GAPDH (Loading Control) antibody

Immunofluorescence of human HeLa cells stained with Hoechst 3342 (Blue) for nucleus staining and monoclonal anti-human GAPDH antibody Cat.-No. AM09367PU (1/500) with Texas Red (Red). Mouse IgG2b AT8G4 Purified Hu E, ICC/IF, WB
50 µl / €230.00
  Acris Antibodies GmbH

TUBB3 / TUBB4 antibody

BM170 TUBB3/TUBB4 antibody staining of Rat Skin Paraffin Section. Mouse IgG1 TU-20 Purified Broad C, E, F, ICC/IF, P, WB
0.2 mg / €350.00
  Acris Antibodies GmbH

TUBB / TUBB5 (N-term) (Loading Control) antibody

Western Blotting analysis (reducing conditions) of HPB-ALL human peripheral blood leukemia cell line. Lane 1: negative control. Lane 2,3,4,5,6: immunostaining with anti-beta-tubulin (TU-06; dilution 0,5 ?g/ml, 1 ?g/ml, 2 ?g/ml, 4 ?g/ml, 5 ?g/ml) Mouse IgM TU-06 Purified All Species ICC/IF, P, WB
0.1 mg / €240.00
  Acris Antibodies GmbH

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