Anti-Sarcomeric Alpha Actinin antibody [EP2529Y]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cardiac muscle tissue labelling Sarcomeric Alpha Actinin with purified ab68167 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human muscle tissue labelling Sarcomeric Alpha Actin with unpurified ab68167 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

Immunohistochemical analysis of formalin fixed paraffin embedded human heart labelling sarcomeric alpha actinin with ab68167 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab68167 anti-sarcomeric alpha actinin antibody [EP2529Y] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Paraffin embedded human skeletal muscle tissue slides were dewaxed, followed by heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, Epitope Retrieval ER2 Solution) for 20 mins. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 mins. The sections were then incubated with ab68167 (1/300) at room temperature for 30 mins, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The sections were counterstained with Hematoxylin. The images were taken with a Leica Aperio AT2.

ab68167 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in IHC-P. The image shows the staining intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

• IP

Unknown

Immunoprecipitation - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

ab68167 (purified) at 1/20 immunoprecipitating Sarcomeric Alpha Actinin in HepG2 whole cell lysate.

Lane 1 (input) : HepG2 whole cell lysate (10μg)

Lane 2 (+) : ab68167 + HepG2 whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab68167 in HepG2 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (ab68167) at 1/20 dilution

Lane 1:

HepG2 whole cell lysate at 10 µg

Lane 2:

ab68167 + HepG2 whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab68167 in HepG2 whole cell lysate

Predicted band size: 103 kDa

Observed band size: 103 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cardiac muscle tissue labelling Sarcomeric Alpha Actinin with purified ab68167 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• WB

Lab

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

Blocking and dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (ab68167) at 1/20000 dilution

All lanes:

Human fetal heart tissue lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 103 kDa

Observed band size: 103 kDa

false

• WB

Lab

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

Blocking and dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (ab68167) at 1/10000 dilution

Lane 1:

Mouse brain tissue lysate at 10 µg

Lane 2:

Rat brain tissue lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 103 kDa

Observed band size: 103 kDa

false

• WB

Lab

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

Blocking and dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (ab68167) at 1/20000 dilution

All lanes:

Mouse heart tissue lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 103 kDa

Observed band size: 103 kDa

false

• WB

Lab

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of mouse brain lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab68167 (1/20,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/100,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab68167 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (ab68167) at 1/20000 dilution

All lanes:

Mouse brain lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 180s

• WB

Unknown

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

All lanes:

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (ab68167) at 1/20000 dilution

Lane 1:

Human skeletal muscle lysate at 10 µg

Lane 2:

Rat heart lysate at 10 µg

Lane 3:

Human heart lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 103 kDa

Observed band size: 103 kDa

false

• WB

CiteAb

Western blot - Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (AB68167)

Sarcomeric Alpha Actinin western blot using anti-Sarcomeric Alpha Actinin antibody [EP2529Y] ab68167. Publication image and figure legend from Hodges, J. L., Vilchez, S. M., et al., 2014, PLoS One, PubMed 25007055.

ab68167 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab68167 please see the product overview.

α-actinin-2 localizes to post-synaptic sites in dendritic spines on hippocampal neurons.A) An anti-α-actinin antibody (ab68167) recognizes α-actinin-2 and not α-actinin-1 or α-actinin-4. CHO-K1 cells were transfected with human α-actinin-1-GFP, α-actinin-2-GFP, or α-actinin-4-GFP. Cells were lysed and immunoblotted for α-actinin-2 and GFP. B) α-Actinin-2 is enriched in hippocampal neurons but not in glia cells or COS-7 cells, which lacks α-actinin-2. Cells were lysed and immunoblotted for α-actinin-2. Actin is the loading control. C) α-Actinin-2 localizes to dendritic spines. Hippocampal neurons were transfected at DIV 17 with GFP (green), and fixed, and immunostained for endogenous α-actinin-2 (magenta) at DIV 21. D) α-Actinin-2 co-localizes with post-synaptic markers, but not with a pre-synaptic marker. Hippocampal neurons were fixed at DIV 16 or 21 and immunostained for endogenous α-actinin-2 (green) and either endogenous synaptophysin, PSD-95, or the NR1 subunit of the NMDA receptor (magenta). E–G) The siRNA is specific for α-actinin-2. Hippocampal neurons were co-transfected at DIV 17 with GFP and either a control empty vector (pSUPER), or a vector containing siRNA against α-actinin-2 (pSUPER-α-actinin-2), or the α-actinin-2 siRNA-containing vector plus a α-actinin-2 vector conferring resistance to RNAi (pSUPER-α-actinin-2+ α-actinin-2-SS). The cells were fixed at DIV 21 and immunostained for endogenous α-actinin-2. Arrows point to the neurons co-expressing GFP and its immunostaining for α-actinin-2. For each condition (55 control cells and 46 α-actinin-2 knockdown cells), the integrated density of the cell body and dendrites were measured from the transfected neuron and adjacent untransfected neuron of the same image and the percent change was plotted, F. Error bars represent SEM. p-values were derived using the paired t-test. G) CHO-K1 cells were co-transfected with GFP, pSUPER or pSUPER-α-actinin-2, plus either α-actinin-2-Flag or α-actinin-2-SS-Flag. Transfection efficiency was close to 100% as >95% of the cells in each condition exhibited GFP fluorescence (data not shown). Cells were lysed 72 hours after transfection and immunoblotted for α-actinin-2 and α-actinin-4. Tubulin is the loading control.

false