Western blot - beta Actin antibody - Loading Control (ab8227)

All lanes : Anti-beta Actin antibody - Loading Control (ab8227) at 1/5000 dilution

Lane 1 : HeLa whole cell extract
Lane 2 : Yeast cell extract
Lane 3 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/2000 dilution

Observed band size : 47 kDa (why is the actual band size different from the predicted?)

Immunocytochemistry/ Immunofluorescence - beta Actin antibody - Loading Control (ab8227)

ICC/IF image of ab8227 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab8227, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue).

Western blot - beta Actin antibody - Loading Control (ab8227)

All lanes : Anti-beta Actin antibody - Loading Control (ab8227) at 1/1000 dilution

Lane 1 : HeLa nuclear lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate
Lane 5 : HEK 293 cell lysate
Lane 6 : HeLa nuclear lysate with beta Actin peptide (ab13772) at 1 µg/ml
Lane 7 : HeLa whole cell lysate with beta Actin peptide (ab13772) at 1 µg/ml
Lane 8 : A431 cell lysate with beta Actin peptide (ab13772) at 1 µg/ml
Lane 9 : Jurkate cell lysate with beta Actin peptide (ab13772) at 1 µg/ml
Lane 10 : HEK 293 cell lysate with beta Actin peptide (ab13772) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Observed band size : 41.7 kDa (why is the actual band size different from the predicted?)


Exposure time : 5 seconds

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - beta Actin antibody - Loading Control (ab8227)

IHC image of beta Actin staining in human liver FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8227, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Immunohistochemistry (Frozen sections) - beta Actin antibody - Loading Control (ab8227)

ab8227 staining beta Actin in human stomach tissue sections by Immunohistochemistry (frozen sections). Tissue was fixed with acetone and then blocked with 5% serum for 1 hour at 23ºC followed by incubation with the primary antibody, at 1µg/ml, for 1 hour at 23ºC. An undiluted HRP-conjugated goat polyclonal was used as secondary antibody.

This image is courtesy of an anonymous abreview.

ELISA - beta Actin antibody - Loading Control (ab8227)

ab8227 used in Direct ELISA in NIH 3T3 murine fibroblasts. Primary antibody used at a 1/1000 dilution for 16 hours at 4°C. The secondary antibody used is an AP-conjugated Goat anti-rabbit used at a 1/2000 dilution. A blocking step was performed using 5% BSA for 1 hour at 23°C.

Image courtesy of an anonymous Abreview.

Immunocytochemistry/ Immunofluorescence - beta Actin antibody - Loading Control (ab8227)

ICC/IF image of ab8227 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8227, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Western blot - beta Actin antibody - Loading Control (ab8227)

All lanes : Anti-beta Actin antibody - Loading Control (ab8227) at 1/1000 dilution

Lane 1 : HeLa cells (Human) at 20 µg
Lane 2 : 3T3 cells (Mouse) at 20 µg
Lane 3 : Fish Liver at 20 µg
Lane 4 : Rabbit Liver at 20 µg
Lane 5 : MDCK cells (Dog) at 20 µg/ml
Lane 6 : EBTr cells (Cow) at 20 µg
Lane 7 : SL-29 cells (Chicken) at 20 µg/ml
Lane 8 : CHO cells (Chinese Hamster) at 20 µg
Lane 9 : Xenopus embryo at 20 µg

Secondary
Rabbit polyclonal Secondary Antibody to Goat IgG - H&L (HRP) (ab6741) at 1/5000 dilution

Observed band size : 41.7 kDa (why is the actual band size different from the predicted?)


Exposure time : 30 seconds