Anti-IKK alpha antibody [Y463] - BSA and Azide free

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-CHUK knockout cells (red line) stained with ab32041. The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab32041, 1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-CHUK knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This antibody can also be used in HAP1 cells fixed with 80% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32041).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

This data was developed using ab32041, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian cancer tissue sections labeling IKK alpha with purified ab32041 at 1/50 dilution (3.92 μg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

This data was developed using ab32041, the same antibody clone in a different buffer formulation. Intracellular Flow Cytometry analysis of Daudi (Human Burkitt's lymphoma lymphoblast) cells labelling IKK alpha with purified ab32041 at 1/20 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IP

Unknown

Immunoprecipitation - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

Purified ab32041 at 1/60 dilution (2μg) immunoprecipitating IKK alpha in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32041 + HeLa whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab169743 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 88 kDa
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32041).

All lanes:

Immunoprecipitation - Anti-IKK alpha antibody [Y463] (<a href='/en-us/products/primary-antibodies/ikk-alpha-antibody-y463-ab32041'>ab32041</a>)

Predicted band size: 84 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

This data was developed using ab32041, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling IKK alpha with purified ab32041 at 1/50 dilution (3.92 μg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

• WB

Unknown

Western blot - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

All lanes:

Western blot - Anti-IKK alpha antibody [Y463] (<a href='/en-us/products/primary-antibodies/ikk-alpha-antibody-y463-ab32041'>ab32041</a>) at 1/10000 dilution

All lanes:

Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysate

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Predicted band size: 84 kDa

false

• WB

Unknown

Western blot - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

All lanes:

Western blot - Anti-IKK alpha antibody [Y463] (<a href='/en-us/products/primary-antibodies/ikk-alpha-antibody-y463-ab32041'>ab32041</a>) at 1/1000 dilution

Lane 1:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 15 µg

Lane 2:

Mouse kidney lysate at 15 µg

Lane 3:

C6 (Rat glial tumor glial cell) whole cell lysate at 15 µg

Lane 4:

Rat kidney lysate at 15 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 1/20000 dilution

Predicted band size: 84 kDa

false

• WB

CiteAb

Western blot - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

Western Blotting using Recombinant Anti-IKK alpha antibody [Y463] - BSA and Azide free, ab169743. Publication image from Clift, D. et al., 2017, Cell, 29153837. Legend direct from paper.

TRIM21 Overexpression Does Not Perturb Cells, Related to Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7(A) Oocytes microinjected with either water (control) or Trim21 mRNA (TRIM21 OE) were imaged for sixteen hours following release from prophase arrest. Microtubules and chromosomes were labeled with mEGFP-Map4 and H2B-mCherry respectively. Time shows hours and minutes (h : min) from nuclear envelope breakdown (NEBD). Scale bars, 10 µm. White dashed line outlines oocyte. Yellow dashed line outlines nucleus.(B–G) Key events in meiosis were quantified. Number of oocytes is specified in brackets. P values were calculated with Fisher’s exact test (B, D, E and G) or Student’s t test (C and F).(H and I) NIH 3T3, NIH 3T3-mCherry-mTRIM21, HEK293T and HEK293T-mCherry-hTRIM21 cell lines were analyzed by flow cytometry. At least 2000 cells were counted for each condition. Percentages correspond to mCherry-positive cells falling within the gate drawn.(J) NIH 3T3 and NIH 3T3-mCherry-mTRIM21 cells (NIH 3T3 mix) or HEK293T and HEK293T-mCherry-hTRIM21 cells (HEK293T mix) were mixed 50 : 50 and percentage mCherry cells analyzed by flow cytometry every 24h for 7 days. At least 10,000 cells counted at each time point. Error bars show s.d. Data from 3 independent replicates.(K–M) Transcriptomes of NIH 3T3-mCherry-mTRIM21 cells were compared to wild-type NIH 3T3 cells by RNA-seq analysis (STAR Methods) following electroporation with PBS (K), BSA (L) or IgG (M). A total of only 8 protein-encoding transcripts were consistently downregulated more than 2-fold in the NIH 3T3-mCherry-mTRIM21 cells (Kdm5d, Ddx3y, Eif2s3y, Uty, Asb4, Fat4, Papss2 and Lama2), although we cannot rule out the possibility that this is an indirect consequence of lentivirus construct integration rather than a direct consequence of TRIM21 overexpression.(N–P) Relative expression levels of genes encoding proteins reported to be ligands of TRIM21. Taken from RNA sequencing data. Irf8 was not detected.See also Tables S1, S2, and S3.(Q) HEK293T and HEK293T-mCherry-hTRIM21 cells were electroporated with PBS or anti-IKKα antibody and whole cell lysates harvested 3 hours later for immunoblotting. IRF-3 protein levels are unaffected by TRIM21 overexpression or activation.

false

• WB

CiteAb

Western blot - Anti-IKK alpha antibody [Y463] - BSA and Azide free (AB169743)

Western Blotting using Recombinant Anti-IKK alpha antibody [Y463] - BSA and Azide free, ab169743. Publication image from Clift, D. et al., 2017, Cell, 29153837. Legend direct from paper.

Time Frame for Protein Depletion by Trim-Away, Related to Figure 5(A–F) NIH 3T3-mCherry-TRIM21 cells were taken up into the Neon Pipette Tip and either electroporated or not (mock). Percentage of dead cells was determined using the trypan blue exclusion assay (A). For long-term analysis of cellular behavior cells were imaged every 15 min for 70 h following electroporation (B-F). For the growth curves cell density was normalized to the initial density once cells had adhered (D). For adherence analysis the percentage of spread cells was quantified in each frame (B and C). Doubling times (E and F) were calculated taking the 5 h time point as reference, because cells were fully adhered then. Data from three independent experiments. Error bars show s.d. P values were calculated with Student’s t test. See also Movie S4.(G and H) HEK293T-mCherry-TRIM21 cells were electroporated with either control IgG, anti-ERK1 or anti-IKKα antibodies and whole cell lysates harvested at the indicated times after electroporation for immunoblotting.(I–M) NIH 3T3-mCherry-TRIM21 cells were electroporated with control IgG or anti-Pericentrin antibody (BD611815) and 3 hours later analyzed for Pericentrin (I and K) or Cdk5rap2 (J and L) localization or cell lysates were immunoblotted for the indicated proteins (M). Merge also shows DNA stained with Hoechst. Number of cells in brackets. Scale bars, 5 µm. Data from two independent experiments. P values were calculated with Fisher’s exact (K) or Student’s t test (L).

false