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phospho-NFKB p65 (Thr435) Polyclonal Antibody

U-937 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20\u2103, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with phospho-NFKB p65 (Thr435) Antibody(bs-5661R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
U-937 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20\u2103, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with phospho-NFKB p65 (Thr435) Antibody(bs-5661R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
Tissue\/cell:Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum,C-0005) at 37\u00b0C for 20 min; Antibody incubation with (phospho-NFKB p65 (Thr435)) polyclonal Antibody, Unconjugated (bs-5661R) 1:100, 90 minutes at 37\u00b0C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37\u00b0C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Tissue\/cell:Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum,C-0005) at 37\u00b0C for 20 min; Antibody incubation with (phospho-NFKB p65 (Thr435)) polyclonal Antibody, Unconjugated (bs-5661R) 1:100, 90 minutes at 37\u00b0C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37\u00b0C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Tissue\/cell:MCF7 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum,C-0005) at 37\u00b0C for 20 min; Antibody incubation with (phospho-NFKB p65 (Thr435)) polyclonal Antibody, Unconjugated (bs-5661R) 1:100, 90 minutes at 37\u00b0C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37\u00b0C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Tissue\/cell:MCF7 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum,C-0005) at 37\u00b0C for 20 min; Antibody incubation with (phospho-NFKB p65 (Thr435)) polyclonal Antibody, Unconjugated (bs-5661R) 1:100, 90 minutes at 37\u00b0C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37\u00b0C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
MCF-7 (H) cells were treated with or without hTNF-\u03b1 (20 ng\/ml) and Calyculin A (100 nM) for 15 min, 25 \u03bcg total protein per lane of cell lysates (see on figure) probed with phospho-NFKB p65 (Thr435) polyclonal antibody, unconjugated (bs-5661R) at 1:1000 dilution and 4\u00b0C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.
MCF-7 (H) cells were treated with or without hTNF-\u03b1 (20 ng\/ml) and Calyculin A (100 nM) for 15 min, 25 \u03bcg total protein per lane of cell lysates (see on figure) probed with phospho-NFKB p65 (Thr435) polyclonal antibody, unconjugated (bs-5661R) at 1:1000 dilution and 4\u00b0C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.
Bioss unconjugated antibody vial

Applications

  • WB
  • ELISA
  • FCM
  • IHC-P
  • IHC-F
  • IF(IHC-P)
  • IF(IHC-F)
  • IF(ICC)

Reactivity

  • Human
  • Mouse

Predicted Reactivity

  • Rat
Overview
Catalog # bs-5661R-100uL
Product Name phospho-NFKB p65 (Thr435) Polyclonal Antibody
Applications
WB ELISA FCM IHC-P IHC-F IF(IHC-P) IF(IHC-F) IF(ICC)
Reactivity Human, Mouse
Predicted Reactivity Rat
Specifications
Conjugation Unconjugated
Host Rabbit
Source KLH conjugated synthetic phosphopeptide derived from human NFKBp65 around the phosphorylation site of Thr435
Modification Site Thr435
Clonality Polyclonal
Isotype IgG
Concentration 1ug/ul
Purification Purified by Protein A.
Storage Buffer 0.01M TBS(pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol.
Storage Condition Shipped at 4℃. Store at -20℃ for one year. Avoid repeated freeze/thaw cycles.
Target
Gene ID 5970
Subcellular location Cytoplasm, Nucleus
Synonyms NFkB-p65Thr435; RELAphospho T435; NFKBp65; NF-Bp65; p65 NF kappaB; p65 NFkB; NFKBp65; RELA; Transcription Factor p65; v rel avian reticuloendotheliosis viral oncogene homolog A nuclear factor of kappa light polypeptide gene enhancer in B cells 3 p65; V Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A; v rel reticuloendotheliosis viral oncogene homolog A avian; v-rel reticuloendotheliosis viral oncogene homolog A; p65NFKB; Avian reticuloendotheliosis viral v rel oncogene homolog A; MGC131774; NFKB 3; NFKB3; Nuclear Factor NF Kappa B p65 Subunit; Nuclear factor of kappa light polypeptide gene enhancer in B cells 3; Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B Cells.
Background NF-kappa-B is a ubiquitous transcription factor involved in several biological processes. It is held in the cytoplasm in an inactive state by specific inhibitors. Upon degradation of the inhibitor, NF-kappa-B moves to the nucleus and activates transcription of specific genes. NF-kappa-B is composed of NFKB1 or NFKB2 bound to either REL, RELA, or RELB. The most abundant form of NF-kappa-B is NFKB1 complexed with the product of this gene, RELA. Four transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011].
Application Dilution
WB 1:300-5000
ELISA 1:500-1000
FCM 1:20-100
IHC-P 1:200-400
IHC-F 1:100-500
IF(IHC-P) 1:50-200
IF(IHC-F) 1:50-200
IF(ICC) 1:50-200

Frequently asked questions

Which applications is phospho-NFKB p65 (Thr435) Polyclonal Antibody (bs-5661R) validated for, and at what dilution?

bs-5661R is validated for WB (1:300-5000); ELISA (1:500-1000); FCM (1:20-100); IHC-P (1:200-400); IHC-F (1:100-500); IF(IHC-P) (1:50-200); IF(IHC-F) (1:50-200); IF(ICC) (1:50-200). These are recommended starting dilutions; optimal dilutions should be determined by the end user.

Which species does phospho-NFKB p65 (Thr435) Polyclonal Antibody (bs-5661R) react with?

Tested reactivity: Human, Mouse. Predicted reactivity (expected from sequence homology but not yet tested): Rat.

How should bs-5661R be stored?

Shipped at 4℃. Store at -20℃ for one year. Avoid repeated freeze/thaw cycles. Storage buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol.

What immunogen was used to raise phospho-NFKB p65 (Thr435) Polyclonal Antibody (bs-5661R)?

KLH conjugated synthetic phosphopeptide derived from human NFKBp65 around the phosphorylation site of Thr435

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