goat anti human tf antibody  (Bio-Rad)

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

doi: 10.1016/j.rpth.2025.103016

Figure Lengend Snippet: Effect of different secondary antibodies on the detection of tissue factor (TF) by Western blotting using the same primary antibody. Cell lysates from HAP-1 wild-type (WT) and HAP-1 TF knockout (KO; both 40 μg) and recombinant TF (rTF; Innovin, 500 pg, Thermo Fisher Scientific, catalog [cat] number 10873566) were run on a gel, and proteins were transferred to a membrane. The blots were blocked with 5% nonfat milk (Walmart, nonfat dry milk powder) in Tris-buffered saline (TBS)-Tween. TF was detected using a goat anti-TF polyclonal antibody (R&D Systems, cat number AF2339) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. Anti-TF antibody binding was detected using either a mouse antigoat horseradish peroxidase (HRP)-linked secondary antibody (Santa Cruz, cat number sc-2354), a rabbit antigoat HRP-linked secondary antibody (Thermo Fisher Scientific, cat number 31402), or a donkey antigoat HRP-linked secondary antibody (Thermo Fisher Scientific, cat number A15999), all diluted 1:5000 in blocking buffer. The membranes were incubated with Clarity Max Western Enhanced Chemiluminescence Substrate (Bio-Rad, cat number 1705062) for 10 seconds. β-Actin was used as a loading control and was detected using a mouse anti–β-actin antibody (Santa Cruz, cat number sc-47778) diluted 1:5000 in 5% bovine serum albumin in TBS-Tween. An HRP-linked horse anti-mouse HRP antibody (Cell Signaling Technologies, cat number 7076) diluted 1:5000 in blocking buffer was used to detect anti–β-actin binding. Clarity Western Enhanced Chemiluminescence Substrate was added for 10 seconds prior to imaging. For imaging, the blot was exposed for 5 seconds (TF) and 10 seconds (β-actin). The blots were visualized using the iBright FL1000 imaging system (Thermo Fisher Scientific). kDa, kilodalton.

Article Snippet: We compared the ability of 4 commercially available anti-human TF antibodies to detect human TF in low-expressing cell lines: rabbit anti-human TF antibody NBP2-15139 (Novus Biologicals), goat anti-human TF antibody AF2339 (R&D Systems), rabbit anti-human TF antibody ab252918 (clone EPR22548-240, designated Abcam 1), and rabbit anti-human TF antibody ab228968 (clone EPR22548-232, designated Abcam 2; ).

Techniques: Western Blot, Knock-Out, Recombinant, Membrane, Saline, Binding Assay, Blocking Assay, Incubation, Control, Imaging

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

doi: 10.1016/j.rpth.2025.103016

Figure Lengend Snippet: Detection of tissue factor (TF) in different cell lines using 4 different anti-TF antibodies. Cell lysates (BxPC-3 [1 μg] and THP-1 control; THP-1 + lipopolysaccharide [LPS]; HAP-1 wild-type [WT]; and HAP-1 TF knockout [KO; all 40 μg]) were run on separate gels and transferred to membranes. TF was detected using 1 of 4 antibodies: (A) Novus NBP2-15139 (rabbit polyclonal), (B) R&D AF2339 (goat polyclonal), and (C) Abcam ab252918 (lot number 1096425-7, designated Abcam 1, rabbit monoclonal) and (D) Abcam ab228968 (designated Abcam 2) diluted 1:1000 in 5% bovine serum albumin in Tris-buffered saline (TBS)-Tween. The blots were blocked with 5% nonfat milk (Walmart, nonfat dry milk powder) in TBS-Tween. The binding of the rabbit anti-TF antibodies was detected using an HRP-linked goat anti-rabbit secondary antibody (Thermo Fisher Scientific, cat number 31460), and the binding of the goat anti-TF antibody was detected using an HRP-linked rabbit antigoat secondary antibody (Thermo Fisher Scientific, cat number 31402). Both secondary antibodies were diluted 1:5000 in 5% milk in TBS-Tween. The membranes were incubated with Clarity Max Western Enhanced Chemiluminescence Substrate (Bio-Rad, cat number 1705062) for 10 seconds. α-Actinin was used as a loading control and detected using a rabbit anti–α-actinin antibody (Cell Signaling Technologies, cat number 3134) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. The binding of the rabbit anti–α-actinin antibody was detected using an HRP-linked goat anti-rabbit secondary (Thermo Fisher Scientific, cat number 31460) diluted 1:5000 in blocking buffer. Clarity Western Enhanced Chemiluminescence Substrate was added for 10 seconds prior to imaging. For imaging, the blots were exposed for 10 seconds (A and B) or 2 seconds for TF detection (C and D) . All blots were exposed for 10 seconds for α-actinin detection. The blots were visualized using the iBright FL1000 imaging system (Thermo Fisher Scientific). For α-actinin, the brightness of the entire blot was adjusted. kDa, kilodalton.

Article Snippet: We compared the ability of 4 commercially available anti-human TF antibodies to detect human TF in low-expressing cell lines: rabbit anti-human TF antibody NBP2-15139 (Novus Biologicals), goat anti-human TF antibody AF2339 (R&D Systems), rabbit anti-human TF antibody ab252918 (clone EPR22548-240, designated Abcam 1), and rabbit anti-human TF antibody ab228968 (clone EPR22548-232, designated Abcam 2; ).

Techniques: Control, Knock-Out, Saline, Binding Assay, Incubation, Western Blot, Blocking Assay, Imaging

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

doi: 10.1016/j.rpth.2025.103016

Figure Lengend Snippet: Effect of deglycosylation on lysates from BxPC-3, HAP-1 wild-type (WT), HAP-1 tissue factor (TF) knockout (KO) cells, and lipopolysaccharide (LPS)-stimulated THP-1 cells. Cell lysates (BxPC-3 [1 μg]; HAP-1 WT and HAP-1 TF KO; and LPS-stimulated THP-1 cells [all 40 μg]) were treated with no enzyme or PNGase F. The lysates from the LPS-stimulated THP-1 cells were run on a separate gel from the other lysates. After running the gels, the proteins were transferred to membranes. The blots were blocked with 5% nonfat milk (Walmart, nonfat dry milk powder) in Tris-buffered saline (TBS)-Tween. TF (glycosylated TF [TF] and deglycosylated TF [dg-TF]) was detected using 1 of 3 antibodies: (A) Novus NBP2-15139, (B) R&D AF2339, and (C) Abcam ab252918 (lot number GR3270793-2, designated Abcam 1) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. The binding of the rabbit anti-TF antibodies was detected using an HRP-linked goat anti-rabbit secondary antibody (Thermo Fisher Scientific, cat number 31460), and the binding of the goat anti-TF antibody was detected using an HRP-linked rabbit antigoat secondary antibody (Thermo Fisher Scientific, cat number 31402). Both secondary antibodies were diluted 1:5000 in 5% milk in TBS-Tween. The membranes were incubated with Clarity Max Western Enhanced Chemiluminescence Substrate (Bio-Rad, cat number 1705062) for 10 seconds. α-Actinin was used as a loading control and detected using a rabbit anti–α-actinin antibody (Cell Signaling Technologies, cat number 3134) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. The binding of the rabbit anti–α-actinin antibody was detected using an HRP-linked goat antir-abbit secondary antibody (Thermo Fisher Scientific, cat number 31460) diluted 1:5000 in blocking buffer. The membranes were incubated with Clarity Western Enhanced Chemiluminescence Substrate for 10 seconds. For imaging, the blots were exposed for 2 seconds for TF detection and 10 seconds for α-actinin detection. The blots were visualized using the iBright FL1000 imaging system (Thermo Fisher Scientific). For α-actinin, the brightness of the entire blot was adjusted. kDa, kilodalton; NS, non-specifc.

Article Snippet: We compared the ability of 4 commercially available anti-human TF antibodies to detect human TF in low-expressing cell lines: rabbit anti-human TF antibody NBP2-15139 (Novus Biologicals), goat anti-human TF antibody AF2339 (R&D Systems), rabbit anti-human TF antibody ab252918 (clone EPR22548-240, designated Abcam 1), and rabbit anti-human TF antibody ab228968 (clone EPR22548-232, designated Abcam 2; ).

Techniques: Knock-Out, Saline, Binding Assay, Incubation, Western Blot, Control, Blocking Assay, Imaging

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

doi: 10.1016/j.rpth.2025.103016

Figure Lengend Snippet: Effect of different secondary antibodies on the detection of tissue factor (TF) by Western blotting using the same primary antibody. Cell lysates from HAP-1 wild-type (WT) and HAP-1 TF knockout (KO; both 40 μg) and recombinant TF (rTF; Innovin, 500 pg, Thermo Fisher Scientific, catalog [cat] number 10873566) were run on a gel, and proteins were transferred to a membrane. The blots were blocked with 5% nonfat milk (Walmart, nonfat dry milk powder) in Tris-buffered saline (TBS)-Tween. TF was detected using a goat anti-TF polyclonal antibody (R&D Systems, cat number AF2339) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. Anti-TF antibody binding was detected using either a mouse antigoat horseradish peroxidase (HRP)-linked secondary antibody (Santa Cruz, cat number sc-2354), a rabbit antigoat HRP-linked secondary antibody (Thermo Fisher Scientific, cat number 31402), or a donkey antigoat HRP-linked secondary antibody (Thermo Fisher Scientific, cat number A15999), all diluted 1:5000 in blocking buffer. The membranes were incubated with Clarity Max Western Enhanced Chemiluminescence Substrate (Bio-Rad, cat number 1705062) for 10 seconds. β-Actin was used as a loading control and was detected using a mouse anti–β-actin antibody (Santa Cruz, cat number sc-47778) diluted 1:5000 in 5% bovine serum albumin in TBS-Tween. An HRP-linked horse anti-mouse HRP antibody (Cell Signaling Technologies, cat number 7076) diluted 1:5000 in blocking buffer was used to detect anti–β-actin binding. Clarity Western Enhanced Chemiluminescence Substrate was added for 10 seconds prior to imaging. For imaging, the blot was exposed for 5 seconds (TF) and 10 seconds (β-actin). The blots were visualized using the iBright FL1000 imaging system (Thermo Fisher Scientific). kDa, kilodalton.

Article Snippet: TF was detected using a goat anti-TF polyclonal antibody (R&D Systems, cat number AF2339) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween.

Techniques: Western Blot, Knock-Out, Recombinant, Membrane, Saline, Binding Assay, Blocking Assay, Incubation, Control, Imaging

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

doi: 10.1016/j.rpth.2025.103016

Figure Lengend Snippet: Detection of tissue factor (TF) in different cell lines using 4 different anti-TF antibodies. Cell lysates (BxPC-3 [1 μg] and THP-1 control; THP-1 + lipopolysaccharide [LPS]; HAP-1 wild-type [WT]; and HAP-1 TF knockout [KO; all 40 μg]) were run on separate gels and transferred to membranes. TF was detected using 1 of 4 antibodies: (A) Novus NBP2-15139 (rabbit polyclonal), (B) R&D AF2339 (goat polyclonal), and (C) Abcam ab252918 (lot number 1096425-7, designated Abcam 1, rabbit monoclonal) and (D) Abcam ab228968 (designated Abcam 2) diluted 1:1000 in 5% bovine serum albumin in Tris-buffered saline (TBS)-Tween. The blots were blocked with 5% nonfat milk (Walmart, nonfat dry milk powder) in TBS-Tween. The binding of the rabbit anti-TF antibodies was detected using an HRP-linked goat anti-rabbit secondary antibody (Thermo Fisher Scientific, cat number 31460), and the binding of the goat anti-TF antibody was detected using an HRP-linked rabbit antigoat secondary antibody (Thermo Fisher Scientific, cat number 31402). Both secondary antibodies were diluted 1:5000 in 5% milk in TBS-Tween. The membranes were incubated with Clarity Max Western Enhanced Chemiluminescence Substrate (Bio-Rad, cat number 1705062) for 10 seconds. α-Actinin was used as a loading control and detected using a rabbit anti–α-actinin antibody (Cell Signaling Technologies, cat number 3134) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. The binding of the rabbit anti–α-actinin antibody was detected using an HRP-linked goat anti-rabbit secondary (Thermo Fisher Scientific, cat number 31460) diluted 1:5000 in blocking buffer. Clarity Western Enhanced Chemiluminescence Substrate was added for 10 seconds prior to imaging. For imaging, the blots were exposed for 10 seconds (A and B) or 2 seconds for TF detection (C and D) . All blots were exposed for 10 seconds for α-actinin detection. The blots were visualized using the iBright FL1000 imaging system (Thermo Fisher Scientific). For α-actinin, the brightness of the entire blot was adjusted. kDa, kilodalton.

Article Snippet: TF was detected using a goat anti-TF polyclonal antibody (R&D Systems, cat number AF2339) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween.

Techniques: Control, Knock-Out, Saline, Binding Assay, Incubation, Western Blot, Blocking Assay, Imaging

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

doi: 10.1016/j.rpth.2025.103016

Figure Lengend Snippet: Effect of different secondary antibodies on the detection of tissue factor (TF) by Western blotting using the same primary antibody. Cell lysates from HAP-1 wild-type (WT) and HAP-1 TF knockout (KO; both 40 μg) and recombinant TF (rTF; Innovin, 500 pg, Thermo Fisher Scientific, catalog [cat] number 10873566) were run on a gel, and proteins were transferred to a membrane. The blots were blocked with 5% nonfat milk (Walmart, nonfat dry milk powder) in Tris-buffered saline (TBS)-Tween. TF was detected using a goat anti-TF polyclonal antibody (R&D Systems, cat number AF2339) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. Anti-TF antibody binding was detected using either a mouse antigoat horseradish peroxidase (HRP)-linked secondary antibody (Santa Cruz, cat number sc-2354), a rabbit antigoat HRP-linked secondary antibody (Thermo Fisher Scientific, cat number 31402), or a donkey antigoat HRP-linked secondary antibody (Thermo Fisher Scientific, cat number A15999), all diluted 1:5000 in blocking buffer. The membranes were incubated with Clarity Max Western Enhanced Chemiluminescence Substrate (Bio-Rad, cat number 1705062) for 10 seconds. β-Actin was used as a loading control and was detected using a mouse anti–β-actin antibody (Santa Cruz, cat number sc-47778) diluted 1:5000 in 5% bovine serum albumin in TBS-Tween. An HRP-linked horse anti-mouse HRP antibody (Cell Signaling Technologies, cat number 7076) diluted 1:5000 in blocking buffer was used to detect anti–β-actin binding. Clarity Western Enhanced Chemiluminescence Substrate was added for 10 seconds prior to imaging. For imaging, the blot was exposed for 5 seconds (TF) and 10 seconds (β-actin). The blots were visualized using the iBright FL1000 imaging system (Thermo Fisher Scientific). kDa, kilodalton.

Article Snippet: We examined the ability of 3 different antibodies to detect TF in low-expressing cell lines: rabbit polyclonal anti-human TF antibody NBP2-15139 (Novus Biologicals), goat polyclonal anti-human TF antibody AF2339 (R&D Systems), and rabbit monoclonal anti-human TF antibody ab252918 (clone EPR22548-240; Abcam).

Techniques: Western Blot, Knock-Out, Recombinant, Membrane, Saline, Binding Assay, Blocking Assay, Incubation, Control, Imaging

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

doi: 10.1016/j.rpth.2025.103016

Figure Lengend Snippet: Detection of tissue factor (TF) in different cell lines using 4 different anti-TF antibodies. Cell lysates (BxPC-3 [1 μg] and THP-1 control; THP-1 + lipopolysaccharide [LPS]; HAP-1 wild-type [WT]; and HAP-1 TF knockout [KO; all 40 μg]) were run on separate gels and transferred to membranes. TF was detected using 1 of 4 antibodies: (A) Novus NBP2-15139 (rabbit polyclonal), (B) R&D AF2339 (goat polyclonal), and (C) Abcam ab252918 (lot number 1096425-7, designated Abcam 1, rabbit monoclonal) and (D) Abcam ab228968 (designated Abcam 2) diluted 1:1000 in 5% bovine serum albumin in Tris-buffered saline (TBS)-Tween. The blots were blocked with 5% nonfat milk (Walmart, nonfat dry milk powder) in TBS-Tween. The binding of the rabbit anti-TF antibodies was detected using an HRP-linked goat anti-rabbit secondary antibody (Thermo Fisher Scientific, cat number 31460), and the binding of the goat anti-TF antibody was detected using an HRP-linked rabbit antigoat secondary antibody (Thermo Fisher Scientific, cat number 31402). Both secondary antibodies were diluted 1:5000 in 5% milk in TBS-Tween. The membranes were incubated with Clarity Max Western Enhanced Chemiluminescence Substrate (Bio-Rad, cat number 1705062) for 10 seconds. α-Actinin was used as a loading control and detected using a rabbit anti–α-actinin antibody (Cell Signaling Technologies, cat number 3134) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. The binding of the rabbit anti–α-actinin antibody was detected using an HRP-linked goat anti-rabbit secondary (Thermo Fisher Scientific, cat number 31460) diluted 1:5000 in blocking buffer. Clarity Western Enhanced Chemiluminescence Substrate was added for 10 seconds prior to imaging. For imaging, the blots were exposed for 10 seconds (A and B) or 2 seconds for TF detection (C and D) . All blots were exposed for 10 seconds for α-actinin detection. The blots were visualized using the iBright FL1000 imaging system (Thermo Fisher Scientific). For α-actinin, the brightness of the entire blot was adjusted. kDa, kilodalton.

Article Snippet: We examined the ability of 3 different antibodies to detect TF in low-expressing cell lines: rabbit polyclonal anti-human TF antibody NBP2-15139 (Novus Biologicals), goat polyclonal anti-human TF antibody AF2339 (R&D Systems), and rabbit monoclonal anti-human TF antibody ab252918 (clone EPR22548-240; Abcam).

Techniques: Control, Knock-Out, Saline, Binding Assay, Incubation, Western Blot, Blocking Assay, Imaging

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

doi: 10.1016/j.rpth.2025.103016

Figure Lengend Snippet: Effect of deglycosylation on lysates from BxPC-3, HAP-1 wild-type (WT), HAP-1 tissue factor (TF) knockout (KO) cells, and lipopolysaccharide (LPS)-stimulated THP-1 cells. Cell lysates (BxPC-3 [1 μg]; HAP-1 WT and HAP-1 TF KO; and LPS-stimulated THP-1 cells [all 40 μg]) were treated with no enzyme or PNGase F. The lysates from the LPS-stimulated THP-1 cells were run on a separate gel from the other lysates. After running the gels, the proteins were transferred to membranes. The blots were blocked with 5% nonfat milk (Walmart, nonfat dry milk powder) in Tris-buffered saline (TBS)-Tween. TF (glycosylated TF [TF] and deglycosylated TF [dg-TF]) was detected using 1 of 3 antibodies: (A) Novus NBP2-15139, (B) R&D AF2339, and (C) Abcam ab252918 (lot number GR3270793-2, designated Abcam 1) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. The binding of the rabbit anti-TF antibodies was detected using an HRP-linked goat anti-rabbit secondary antibody (Thermo Fisher Scientific, cat number 31460), and the binding of the goat anti-TF antibody was detected using an HRP-linked rabbit antigoat secondary antibody (Thermo Fisher Scientific, cat number 31402). Both secondary antibodies were diluted 1:5000 in 5% milk in TBS-Tween. The membranes were incubated with Clarity Max Western Enhanced Chemiluminescence Substrate (Bio-Rad, cat number 1705062) for 10 seconds. α-Actinin was used as a loading control and detected using a rabbit anti–α-actinin antibody (Cell Signaling Technologies, cat number 3134) diluted 1:1000 in 5% bovine serum albumin in TBS-Tween. The binding of the rabbit anti–α-actinin antibody was detected using an HRP-linked goat antir-abbit secondary antibody (Thermo Fisher Scientific, cat number 31460) diluted 1:5000 in blocking buffer. The membranes were incubated with Clarity Western Enhanced Chemiluminescence Substrate for 10 seconds. For imaging, the blots were exposed for 2 seconds for TF detection and 10 seconds for α-actinin detection. The blots were visualized using the iBright FL1000 imaging system (Thermo Fisher Scientific). For α-actinin, the brightness of the entire blot was adjusted. kDa, kilodalton; NS, non-specifc.

Article Snippet: We examined the ability of 3 different antibodies to detect TF in low-expressing cell lines: rabbit polyclonal anti-human TF antibody NBP2-15139 (Novus Biologicals), goat polyclonal anti-human TF antibody AF2339 (R&D Systems), and rabbit monoclonal anti-human TF antibody ab252918 (clone EPR22548-240; Abcam).

Techniques: Knock-Out, Saline, Binding Assay, Incubation, Western Blot, Control, Blocking Assay, Imaging