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Synthetic peptide corresponding to beta Actin aa 1-14 (N terminal) conjugated to Keyhole Limpet Haemocyanin (KLH). Slightly modified ß-cytoplasmic actin N-terminal peptide, Ac-Asp-Asp-Asp-Ile-Ala-Al?a-Leu-Val-Ile-Asp-Asn-Gl y?-Ser-Gly-Lys, conjugated to KLH.
Immunofluoresence staining of chicken gizzard ultra-thin sections (North et al. J. Cell Sci. 107:445-455 (1994)) labels the dense bodies, longitudinal channels and membrane associated dense-plaque.
This product was changed from ascites to tissue culture supernatant on 31st January 2017. The following lots are from ascites and are still in stock on 31st January 2017- GR231981, GR247612 and GR181659. Lot numbers higher than GR247612 will be from tissue culture supernatant. Please note that the dilutions may need to be adjusted accordingly.
Our Abpromise guarantee covers the use of ab6276 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 5 µg/ml.|
|IHC-FoFr||Use at an assay dependent concentration.|
|ICC||Use at an assay dependent concentration.|
|IHC-P||Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|IHC-Fr||Use at an assay dependent concentration.|
|Indirect ELISA||Use at an assay dependent concentration.|
|WB||1/5000 - 1/16000. Predicted molecular weight: 42 kDa.|
|ELISA||Use at an assay dependent concentration.|
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Beta actin knockout HAP1 cell lysate (20 µg)
Lanes 1 and 2: Merged signal (red and green). Green - ab6276 observed at 42 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab6276 was shown to specifically react with beta actin when beta actin knockout samples were used. Wild-type and beta actin knockout samples were subjected to SDS-PAGE. ab6276 and ab181602 (loading control to GAPDH) were diluted 1/5000 and 1/10 000 and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
ab6276 staining beta Actin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab6276 at a working concentration of 5μg/ml and ab190573, Rabbit monoclonal [EP1332Y] to alpha Tubulin (Alexa Fluor® 647, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse AlexaFluor® 488 (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Immunohistochemical frozen analysis of acetone-fixed human stomach tissue labeling beta Actin with ab6276 at 1/5000 dilution,followed by secondary antibody.
This image is courtesy of an anonymous Abreview.
Detection: Near-Infrared Fluorescence Imaging.
This image is courtesy of David Grimm, Yale University, USA.
MDCK cells induced with increasing amounts of doxycycline to control expression of the gene of interest. All cells were normalized for loading with an albumin protein standard assay. Anti-beta actin (ab6276) was used at a concentration of 1:5000 in a milk blocking solution. B-actin blotting confirms the albumin assay in showing that an equal amount of lysate was loaded in each lane.
MDCK cells induced with increasing amounts of doxycycline to control expression of the gene of interest. All cells were normalized for loading with an albumin protein standard asssay. Anti-beta actin (ab6276) was used at a concentration of 1:5000 in a milk blocking solution. B-actin blotting confirms the albumin assay in showing that an equal amount of lysate was loaded in each lane.
ab6276 staining beat actin in rat hypothalamus primary cells by ICC/IF. The cells were formaldehyde fixed, permeabilized in 0.5% Triton X-100 and blocked in 5% serum for 20 minutes at 25°C. The primary antibody was diluted 1/1000 in PBS (0.1% Triton X-100, 1% goat serum) and incubated with sample for 16 hours at 4°C. An Alexa Fluor® 546 conjugated goat polyclonal to mouse IgG1, diluted 1/500 was used as secondary.
This image is courtesy of an anonymous abreview.
Reduced, denatured 4-12% Bolt Bis-Tris Plus gel.
Blocking: 100% Li-Cor Odyssey Blocking Buffer (TBS).
This image is courtesy of an Abreview submitted by Dr Mark Elliott
Western Blot of ab6276 (used as loading control) with whole tissue lysate of grey matter from BA20 (temporal cortex). Ab6276 was diluted 1/50000 and incubated with the sample for 16 hours at 4°C. 5 µg of lysate was loaded onto the gel, which was blocked with 5% milk for 1 hour at 20°C. An Alexa Fluor® 680 conjugated goat anti-mouse antibody, diluted 1/5000, was used as the secondary.
Bands below actin in image are synaptophysin (SYN).
ab6276 at 1/10000 staining mouse brain tissue sections by IHC-P. The mouse brain was immersion-fixed in 4% Formalin/PBS for 2 days, then bisected mid-saggitally. The tissue was further fixed in same solution for a further 3 days. Both halves were processed to pwax on a dip-n-dunk tissue processor using a 20hr-cycle. Sections were cut at 5microns,floated out on water at 47°C, mounted on Superfrost Plus slides and dried/melted for 24hrs in a 62°C oven.
The whole brain shows positivity but there are areas/cells that are enriched. Note that the axonal tracks are negatively stained at this dilution factor, yet some cells within those tracks are still strongly positive (many similar cells outside of the tracks are also positive).
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"