MA5-11866

antibody from Invitrogen Antibodies

Targeting: ACTG2 ACTA3, ACTL3, ACTSG

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Data presented on provider website)

Immunohistochemistry (Recommended by provider)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

MA5-11866

Provider

Invitrogen Antibodies

Product name

Actin Monoclonal Antibody (ACTN05 (C4)), Biotin

Antibody type

Monoclonal

Antigen

Other

Description

This antibody reacts with all six known vertebrate isoforms of actin (MW ~42 kDa), as well as cytoplasmic actin beta and actin gamma. This antibody is highly recommended for monitoring total protein load on Western blots. Staining of formalin-fixed, paraffin-embedded tissues requires boiling tissue sections in 10mM citrate buffer, pH 6.0 for 10-20 minutes, followed by cooling at room temperature for 20 minutes. Suggested positive controls are HeLa cells and skeletal muscle.

Reactivity

Human, Mouse, Rat, Bovine, Canine, Chicken/Avian, Porcine, Rabbit

Host

Mouse

Conjugate

Biotin

Isotype

IgG

Antibody clone number

ACTN05 (C4)

Vial size

500 µL

Concentration

0.2 mg/mL

Storage

4° C

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of actin was performed by loading 25 µg of various whole cell lysates and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for at least 1 hour. The membrane was probed with a biotinylated pan actin monoclonal antibody (Product # MA5-11866) at a dilution of 1:6400 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with HRP-conjugated Streptavidin (Product # 21126) at a dilution of 1:40,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot of Actin pan using Actin pan Monoclonal Antibody (Product # MA5-11866) on HeLa Cells.

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), A549 (Lane 2), COS-7 (Lane 3), MDCK (Lane 4), NIH/3T3 (Lane 5), HT-29 (Lane 6), PC-12 (Lane 7), Neuro-2a (Lane 8), tissue extracts of Mouse Brain (Lane 9) and Rat Skeletal Muscle (Lane 10). The blot was probed with Actin Monoclonal Antibody (ACTN05 (C4)), Biotin (Product # MA5-11866, 1:5000 dilution) and detected by chemiluminescence using Poly-HRP Streptavidin (Product # N200, 1:10000 dilution). A 42 kDa band corresponding to Actin was observed across the cell lines and tissues tested.

Conjugate

Biotin

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of Actin pan (green) showing staining in the cytoplasm of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with an Actin pan monoclonal antibody (Product # MA5-11869) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of Actin pan (green) showing staining in the cytoplasm of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with an Actin pan monoclonal antibody (Product # MA5-11869) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of Actin pan (green) showing staining in the cytoplasm of T-47D cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with an Actin pan monoclonal antibody (Product # MA5-11869) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of actin (green) in COS7 cells. Cells were fixed and permeabilized with ice-cold methanol for 10 minutes at room temperature, and blocked with 0.3% BSA in PBS for 1 hour at room temperature. Cells were probed with a biotinylated pan actin monoclonal antibody (Product # MA5-11866) at a dilution of 1:50 (right panel) or incubated in blocking buffer as a negative control (left panel) overnight at 4°C, washed with PBS, and incubated with DyLight 488-conjugated Streptavidin (Product # 21832) at a dilution of 1:250 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI (Product # 46190). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of actin (green) in COS7 cells. Cells were fixed and permeabilized with ice-cold methanol for 10 minutes at room temperature, and blocked with 0.3% BSA in PBS for 1 hour at room temperature. Cells were probed with a biotinylated pan actin monoclonal antibody (Product # MA5-11866) at a dilution of 1:50 (right panel) or incubated in blocking buffer as a negative control (left panel) overnight at 4°C, washed with PBS, and incubated with DyLight 488-conjugated Streptavidin (Product # 21832) at a dilution of 1:250 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI (Product # 46190). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Conjugate

Biotin

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoprecipitation of Actin pan using Actin pan Monoclonal Antibody (Product # MA5-11866) on denatured Human HeLa Cells.

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

FIGURE 3: Protein degradation defects in the hac1Deltalpl1Delta mutant. (A) Levels of ubiquitin conjugates in whole-cell extracts of wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains, as determined by SDS-PAGE followed by immunoblot with anti-ubiquitin antibody (top) or anti-Pgk1 antibody (bottom; loading control). Experiment was performed at 37degC. (B) Cycloheximide chase analysis of CPY* turnover in wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains, as determined by SDS-PAGE followed by immunoblot with anti-HA antibody (top) or anti-actin antibody (bottom; loading control). Experiment was performed at 37degC. See Supplemental Figure S1 for quantitation. (C) Cycloheximide chase analysis of turnover of the cytoplasmic proteasome substrate Delta2-GFP, as determined by SDS-PAGE followed by immunoblot with anti-HA antibody (top) or anti-Pgk1 antibody (bottom; loading control). Experiment was performed at 37degC. (D) Growth of wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains expressing galactose-inducible wild-type STE6 or the misfolded mutant ste6-Q1249X. Cells were spotted in threefold serial dilutions and cultured for 4 d at 35degC. (E) Levels of phosphorylated eIF2alpha (Ser-51) in whole-cell extracts of wild-type, lpl1Delta , hac1Delta , and hac1Deltalpl1Delta strains treated with sodium arsenite (0.2 mM), as determined by SDS-PAGE followed by immunoblot with anti-phospho-eIF2alpha antibody (top) or anti-Pgk1 antibody (bottom

Conjugate

Biotin

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3 Differences of RNA-binding motif protein 3 (RBM3) protein expression in various human breast cancer cell lines by western blot analysis. RBM3 protein is markedly increased in MCF-7 and T47D (luminal A subtype) and ZR-75-1 (luminal B subtype). On the other hand, RBM3 protein is significantly reduced in HCC1954 (HER2-enriched type) and BT-20 (basal-like subtype).

Conjugate

Biotin