Loading controls for western blots grade 6 electricity

Figure 1. Loading controls: A, beta-actin. This image is adapted from Figure 1 in [ 1]. B, tubulin. This image is adapted from Figure 1 in [ 2]. C, GAPDH. This image is adapted from Figure 2S in [ 3]. D, lamin B. This image is adapted from Figure 1 in [ 2]. E, HSC70. This image is adapted from Figure 2 in [ 4]. F, vinculin. This image is adapted from Figure 3 in [ 5].

• the same amount of protein sample is loaded in each lane,• proteins are transferred from the gel to the membrane with equal efficiency among different lanes,• antibody incubation (for primary antibody, and if necessary, secondary antibody), and signal detection are uniform across different lanes.

The signals from loading controls are typically used to normalize the signals from the proteins of interest. It should be emphasized that the blotting with control protein antibody should be done on the same blot as the experimental antibody.

Labome surveyed 112 publications with loading controls in Western blots. The results indicate that actin (specifically beta actin) is the most commonly used control. Figure 1 displays the Western blot images compiled from some of those publications. Other interesting observations from this survey are discussed in this article later.

Subtypes of alpha tubulins include 1a (TUBA1A), 1b (TUBA1B), 1c (TUBA1C), 3c (TUBA3C), 3d (TUBA3D), 3e (TUBA3E), 4a (TUBA4A), and 8 (TUBA8). Classes I (TUBB), IIa (TUBB2A), IIb (TUBB2B), III (TUBB3), IVa (TUBB4A), IVb (TUBB4B), V (TUBB6), VI (TUBB1), and VIII (TUBB8) are the subtypes of beta tubulins.

Gamma tubulins, gamma 1(TUBG1) and 2 (TUBG2), are involved in the nucleation and polar orientation of microtubules, and are present in centrosomes and spindle pole bodies. Delta (TUBD1) and epsilon (TUBE1) tubulins tend to localize at centrioles and are the structural components of mitotic spindles.

Tubulins are conserved across species. For example, human tubulin alpha 1a with 451 amino acids share 74% identity with its yeast homolog Tub3p with 445 amino acids. Subtypes of alpha, beta, or gamma are very similar as well. Human alpha tubulins share more than 90% identity with each other. Different types of tubulins share significant homology as well. Human alpha tubulins share 40% identity with human beta tubulins.

Tubulins are pharmacological targets for several groups of drugs, like anti-cancer drugs Taxol, Tesetaxel, vinblastine, and vincristine, anti-gout agent colchicine, and anti-fungal drug griseofulvin. These drugs affect tubulin expression in vitro and in vivo.

Santa Cruz goat polyclonal anti-actin antibodies were used to study the DNA damage signaling in A-T-like disease and Nijmegen breakage syndrome [ 23], in articular chondrocytes [ 24], in SAOS-2 cells (ATCC HTB-85) [ 25], and in HeLa cells [ 26]. Its rabbit polyclonal anti-actin antibody was used in experiments with porcine renal epithelial LLC-PK1 cells [ 27]. Santa Cruz Biotechnology anti-beta-actin antibodies served as loading controls in HUVEC samples [ 28], in the nuclear protein extracts isolated from the murine cardiomyocytes [ 29], in mouse rhabdomyosarcoma cells and RMS772 cells [ 30], in cell extracts [ 31], in mutant and normal B lymphocytes [ 32], in 293T cells [ 33], and in retinal pigmented epithelial cells [ 34].

Actin antibodies from EMD Millipore/Chemicon were used in lysate samples prepared from DAT-KO mice with LiCl, vehicle or alpha MPT injection [ 35], from HT-29 and SK-OV-3 cells treated with NMT1-1 siRNA or NMT2-4 siRNA [ 36], in mouse tissue lysate, C2C12 cell lysate, and HEK293T cell lysate [ 37].

Sigma anti-actin antibodies were used in Western blots for the detection of p53, WAF1, and MDM2 [ 41], in Hela, wild-type H2AX (H2AXflox/flox), and H2AX-deficient ES cells [ 42], in a B-lymphoid precursor cell line [ 43], of TRIM5 proteins in rhesus monkeys [ 44], in U937 cells [ 45], in fibroblasts and surgical brain samples [ 46], in murine bone marrow cells [ 47], and in migrating T cells [ 48]. Sigma anti alpha-actin antibody was used in Western blots as a loading control in HeLa cells tranfected with PKD RNAi, Hsp27 RNAi, and vector control [ 49]. Sigma anti beta-actin antibodies were used to identify transcription factors potentially important for mammalian neuronal differentiation [ 50], in hepatocytes [ 51], in human coronary artery endothelial cells [ 52], in HeLa cells transfected with Luc siRNA or NudC siRNA [ 53], in NCI-H460 and NCI-H1299 cells [ 54], in human astrocytoma cell line 1321N1 [ 55], in TAK1+/+ and TAK1-/- MEFs and HeLa S3 cells [ 56], in both nucleus and cytoplasm of HeLa cells [ 57], in wild-type Jurkat and Jurkat cells stably transfected with Bcl-xL, Bcl-2 or vector control cells [ 58], in splenic B lymphocytes from p85-deficient mice and the corresponding wild-type mice [ 59], in mouse collagen-adherent FN-/- cells [ 60], in K562-R, LAMA-R, and their parent cells treated with 1 M imatinib mesylate [ 61], in ChIP experiments (A5316) [ 62], and in Drosophila samples [ 63]. Sigma actin antibodies were also cited among other articles [ 64- 69].

Roche Molecular Biochemicals anti-actin (C4) antibody were used in [ 70, 71]. Cell Signalling anti-actin antibody was used to study the role of ING1a in senescence [ 72]. Cytoskeleton goat anti-human actin antibody was used in Western blots to study the effect of P2Y2 nucleotide receptors on alpha-secretase-dependent amyloid precursor protein processing [ 73].

Santa Cruz Biotechnology anti-alpha-tubulin antibody was used in human fibroblast GM00637 cells with MT-I/II or MT-III expression [ 75], in transfected U937 cells [ 76], in mouse tumor lysate and to study the role of Che-1 in activation of p21WAF1/Cip1 [ 77]. Santa Cruz Biotechnology anti-beta-tubulin antibody was used in 786-O cells [ 39] and in K562 cells [ 78]. Santa Cruz anti-tubulin antibody (TU-02) was used in Western blots to study the speicficity of the RECQL4 antibodies [ 79].

Sigma anti-alpha tubulin antibodies were used as loading controls in LNCaP, PC3M cells [ 80], to study adenomatous polyposis coli down-regulation by the ubiquitin-proteasome pathway [ 81], in COS cells expressing MuSK protein [ 82], in human umbilical vascular endothelial cell [ 83], in T24 and Hela cells [ 84]. Sigma monoclonal anti-beta-Tubulin antibody was used in human head and neck carcinoma cell lines A253 [ 85], in fibroblast cultures from spinal muscular atrophy (SMA) patients [ 86], and in MCF-7 cells, HCT116 p53+/+ cells, RKO cells, p53 wild-type M7TS90 and p53-null M7TS90-E6 cells [ 87]. Sigma anti-tubulin monoclonal antibody (clone DM1A) was used in embryonic and adult brain samples [ 88], and in NIH 3T3 cells transiently transfected with vector, RACK1, or mutant RACK1 [ 89].

BD PharMingen anti-beta-tubulin antibody was used in Western blots of samples from HeLa Tet-on cells, HeLa Tet-on-inducible FOXO4 stable cells (15-14), and BJAB cells or RCC4 cell stably transfected with a wild-type human VHL expression plasmid [ 90]. Amersham Biosciences anti-alpha-tubulin antibody was used in Jurkat cells [ 91]. Oncogene Science monoclonal anti-tubulin antibody was used in Jurkat cells as well [ 92].