VHH Single Domain Anti-Rabbit IgG Fc (HRP)

• WB

AbReview45923****

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

Blocking Solution and Diluent 5% milk in TBS

All lanes:

Western blot - Anti-KAT3B / p300 antibody (<a href='/en-us/products/primary-antibodies/kat3b-p300-antibody-ab10485'>ab10485</a>) at 1/5000 dilution

Lane 1:

Human MCF-7 Cell Lysate at 50000 Cells

Lane 2:

MDA-MB-231 Cell Lysate at 50000 Cells

Secondary

All lanes:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866)

Predicted band size: 264 kDa

true

Exposure time: 3min

This image is courtesy of an anonymous Abreview

• WB

Lab

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 1% Milk before being incubated with ab70358 overnight at 4°C. Antibody binding was detected using an anti-rabbit VHH single domain antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

All lanes:

Western blot - Anti-PCK1/PEPC antibody (<a href='/en-us/products/primary-antibodies/pck1-pepc-antibody-ab70358'>ab70358</a>) at 1 µg

Lane 1:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Liver (Rat) Tissue Lysate at 10 µg

Lane 3:

Liver (Mouse) Tissue Lysate at 10 µg

Lane 4:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg

Secondary

All lanes:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) at 1/50000 dilution

Predicted band size: 69 kDa

Observed band size: 32 kDa,45 kDa,69 kDa

true

Exposure time: 20s

• WB

Lab

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab25949 overnight at 4°C. Antibody binding was detected using VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866), and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-RBPJK antibody (<a href='/en-us/products/primary-antibodies/rbpjk-antibody-ab25949'>ab25949</a>) at 1 µg/mL

All lanes:

Human pancreas tissue lysate - total protein (<a href='/en-us/products/unavailable/human-pancreas-tissue-lysate-total-protein-ab29816'>ab29816</a>) at 20 µg

Secondary

All lanes:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) at 1/50000 dilution

Predicted band size: 56 kDa

Observed band size: 56 kDa,98 kDa

true

Exposure time: 16min

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

IHC image of VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) staining in formalin fixed paraffin embedded normal human colon tissue section.

The section was dewaxed and then pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked using in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1h at room temperature. The section was then incubated with rabbit polyclonal antibody to Ki67 (ab15580, 0.1μg/ml) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. Endogenous peroxidases were quenched using 1.6% (v/v) hydrogen peroxide in TBS containing 0.025% (v/v) Triton X-100 for 30 minutes at room temperature, with agitation. The secondary antibody, VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866, 0.025μg/ml) was then applied for 1 hour at room temperature in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA before being developed for 10 minutes at room temperature using Steady DAB/Plus (ab103723). The section was then counterstained with hematoxylin and mounted with DPX.

The negative control (secondary antibody only, no primary) inset shows no staining, demonstrating secondary antibody specificity.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

Top left : IHC image of VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) staining in formalin fixed paraffin embedded normal human colon tissue section.

The section was dewaxed and then pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked using in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1h at room temperature. The section was then incubated with rabbit polyclonal antibody to Ki67 (ab15580, 0.1μg/ml) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. Endogenous peroxidases were quenched using 1.6% (v/v) hydrogen peroxide in TBS containing 0.025% (v/v) Triton X-100 for 30 minutes at room temperature, with agitation. The secondary antibody, VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866, 0.1μg/ml) was then applied for 1 hour at room temperature in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA before being developed for 10 minutes at room temperature using Steady DAB/Plus (ab103723). The section was then counterstained with hematoxylin and mounted with DPX.

Top right : this image shares the same experimental design parameters, except the secondary antibody was ab97051, goat anti-rabbit IgG H&L (HRP) (0.1μg/ml). This demonstrates the improved definition of staining given by VHH Single Domain Antibodies over conventional secondaries.

Bottom right : this image shares the same experimental design parameters but is a negative control (no primary antibody) for ab97051, demonstrating specificity of the goat anti-rabbit secondary antibody.

Bottom left : this image shares the same experimental design parameters but is a negative control (no primary antibody) for ab191866, demonstrating the specificity of the VHH-Single Domain Antibody.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

IHC image of VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) staining in formalin fixed paraffin embedded normal human colon tissue section.

The section was dewaxed and then pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked using in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1h at room temperature. The section was then incubated with rabbit polyclonal antibody to beta tubulin (ab6046, 0.5μg/ml) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. Endogenous peroxidases were quenched using 1.6% (v/v) hydrogen peroxide in TBS containing 0.025% (v/v) Triton X-100 for 30 minutes at room temperature, with agitation. The secondary antibody, VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866, 0.125μg/ml) was then applied for 1 hour at room temperature in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA before being developed for 10 minutes at room temperature using Steady DAB/Plus (ab103723). The section was then counterstained with hematoxylin and mounted with DPX.

The negative control (secondary antibody only, no primary) inset shows no staining, demonstrating secondary antibody specificity.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

IHC image of VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) staining in formalin fixed paraffin embedded normal human colon tissue section.

The section was dewaxed and then pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked using in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1h at room temperature. The section was then incubated with rabbit polyclonal antibody to VDAC1 (ab15895, 1/1000 dilution) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. Endogenous peroxidases were quenched using 1.6% (v/v) hydrogen peroxide in TBS containing 0.025% (v/v) Triton X-100 for 30 minutes at room temperature, with agitation. The secondary antibody, VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866, 0.125μg/ml) was then applied for 1 hour at room temperature in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA before being developed for 10 minutes at room temperature using Steady DAB/Plus (ab103723). The section was then counterstained with hematoxylin and mounted with DPX.

The negative control (secondary antibody only, no primary) insert shows no staining, demonstrating secondary antibody specificity.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

IHC image of VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) staining in formalin fixed paraffin embedded normal human cerebellum tissue section.

The section was dewaxed and then pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. Non-specific protein-protein interactions were then blocked using in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1h at room temperature. The section was then incubated with rabbit monoclonal antibody [EPR12763] to NeuN (ab177487, 0.1μg/ml) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. Endogenous peroxidases were quenched using 1.6% (v/v) hydrogen peroxide in TBS containing 0.025% (v/v) Triton X-100 for 30 minutes at room temperature, with agitation. The secondary antibody, VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866, 1.0μg/ml) was then applied for 1 hour at room temperature in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA before being developed for 10 minutes at room temperature using Steady DAB/Plus (ab103723). The section was then counterstained with hematoxylin and mounted with DPX.

The negative control (secondary antibody only, no primary) inset shows no staining, demonstrating secondary antibody specificity.

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

• WB

Lab

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab5823 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Histone H4 (symmetric di methyl R3) antibody (<a href='/en-us/products/primary-antibodies/histone-h4-symmetric-di-methyl-r3-antibody-ab5823'>ab5823</a>) at 1 µg

All lanes:

Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

Secondary

All lanes:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) at 1/50000 dilution

Predicted band size: 11 kDa

Observed band size: 14 kDa

true

Exposure time: 3min

• WB

Lab

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab45145 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

Lanes 1 - 2:

Western blot - Anti-Aurora B antibody [EP1009Y] (<a href='/en-us/products/primary-antibodies/aurora-b-antibody-ep1009y-ab45145'>ab45145</a>) at 1/1000 dilution

Lanes 3 - 4:

Western blot - Anti-Aurora B antibody [EP1009Y] (<a href='/en-us/products/primary-antibodies/aurora-b-antibody-ep1009y-ab45145'>ab45145</a>) at 1/5000 dilution

Lanes 5 - 6:

Western blot - Anti-Aurora B antibody [EP1009Y] (<a href='/en-us/products/primary-antibodies/aurora-b-antibody-ep1009y-ab45145'>ab45145</a>) at 1/10000 dilution

Lanes 7 - 8:

Western blot - Anti-Aurora B antibody [EP1009Y] (<a href='/en-us/products/primary-antibodies/aurora-b-antibody-ep1009y-ab45145'>ab45145</a>) at 1/50000 dilution

Lanes 9 - 10:

Western blot - Anti-Aurora B antibody [EP1009Y] (<a href='/en-us/products/primary-antibodies/aurora-b-antibody-ep1009y-ab45145'>ab45145</a>) at 1/75000 dilution

Lanes 1, 3, 5, 7 and 9:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lanes 2, 4, 6, 8 and 10:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Nocodozole Stimulated at 10 µg

Secondary

All lanes:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) at 1/10000 dilution

Predicted band size: 39 kDa

Observed band size: 39 kDa

true

Exposure time: 8min

• WB

Lab

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab24193 overnight at 4°C. Antibody binding was detected using VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866), and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-n-Myc/MYCN antibody (<a href='/en-us/products/primary-antibodies/n-myc-mycn-antibody-ab24193'>ab24193</a>) at 1 µg/mL

Lane 1:

Heart (Human) Whole Cell Lysate - fetal normal tissue at 10 µg

Lane 2:

Heart (Mouse) Tissue Lysate at 10 µg

Lane 3:

Skeletal Muscle (Mouse) Tissue Lysate at 10 µg

Secondary

All lanes:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) at 1/50000 dilution

Predicted band size: 50 kDa

Observed band size: 190 kDa,49 kDa

true

Exposure time: 30s

• WB

Lab

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

All lanes:

Western blot - Anti-GAPDH antibody - Loading Control (<a href='/en-us/products/primary-antibodies/gapdh-antibody-loading-control-ab37168'>ab37168</a>) at 1 µg/mL

Lanes 1 and 3:

HeLa Whole Cell Lysate at 10 µg

Lanes 2 and 4:

Jurkat Whole Cell Lysate at 10 µg

Secondary

Lanes 1 - 2:

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 0.02 µg/mL

Lanes 3 - 4:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) at 0.02 µg/mL

Predicted band size: 11 kDa,54 kDa

true

• WB

Lab

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

All lanes:

Western blot - Anti-NRG1 type III antibody (<a href='/en-us/products/primary-antibodies/nrg1-type-iii-antibody-ab23248'>ab23248</a>) at 1 µg/mL

Lanes 1 and 3:

Mouse Brain Tissue Lysate at 10 µg

Lanes 2 and 4:

Rat Brain Tissue Lysate at 10 µg

Secondary

Lanes 1 - 2:

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 0.05 µg/mL

Lanes 3 - 4:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) at 0.05 µg/mL

Predicted band size: 11 kDa

true

• WB

Lab

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (AB191866)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab16044 overnight at 4°C. Antibody binding was detected using VHH Single Domain Anti-Rabbit IgG Fc (HRP), and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-CD3 antibody (<a href='/en-us/products/primary-antibodies/cd3-antibody-ab16044'>ab16044</a>) at 1 µg/mL

Lane 1:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 2:

Thymus (Mouse) Tissue Lysate at 20 µg

Lane 3:

Thymus (Rat) Tissue Lysate at 20 µg

Secondary

All lanes:

Western blot - VHH Single Domain Anti-Rabbit IgG Fc (HRP) (ab191866) at 1/50000 dilution

Predicted band size: 19 kDa

Observed band size: 18 kDa,23 kDa,48 kDa,62 kDa

true

Exposure time: 8min