Custom solutions & partnerships Custom antibody development and commercial partnerships to advance your diagnostic and therapeutic discovery. Keep up to date with the latest events Full event breakdown with abstracts, speakers, registration and more View global event calendar Specificity The antibody used for conjugation reacts with rabbit immunoglobulins of all classes.Cross-reactions as determined by ELISA for the unconjugated antibody (ab182016): Mouse IgG, rat IgG, and chicken IgY, less than 2%. Human IgG, less than 7%. Storage instructions Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. Store In the Dark. Storage buffer pH: 7.40Preservative: 0.1% Proclin 300 SolutionConstituents: PBS, 1% BSA, 30% Glycerol (glycerin, glycerine) Purification notes This antibody was isolated by affinity chromatography using antigen coupled to agarose beads and conjugated to Horse Radish Peroxidase (HRP). The Abpromise guarantee Our Abpromise guarantee covers the use of ab205718 in the following tested applications. The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user. IP Use at an assay dependent concentration. Notes IHC-P1/2000 - 1/50000. WB1/2000 - 1/50000. ELISA1/5000 - 1/20000. IPUse at an assay dependent concentration. Images Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab205718) All lanes : Anti-beta Actin antibody (ab8227) at 1 µg/mlLane 1 : Liver (Human) Tissue LysateLane 2 : Liver (Mouse) Tissue LysateLane 3 : Liver (Rat) Tissue LysateLane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell LysateLane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell LysateLysates/proteins at 10 µg per lane.SecondaryAll lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab205718) at 1/50000 dilutionDeveloped using the ECL technique.Performed under reducing conditions.Observed band size: 42 kDa why is the actual band size different from the predicted?Exposure time: 30 secondsThis 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 ab8227 overnight at 4 C. Antibody binding was detected using ab205718, and visualised using ECL development solution ab133406. IHC image of histone H4 staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4 C with ab177840 at 1/1000 dilution. An HRP-conjugated secondary (Ab205718, 1/20000 dilution) was used to detect the primary for 1hr at room temperature. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre All lanes : Anti-beta Actin antibody (ab8227) at 1 µg/mlLane 1 : Liver (Mouse) Tissue LysateLane 2 : Liver (Rat) Tissue LysateLysates/proteins at 10 µg per lane.SecondaryAll lanes : ab205718 (Left Image) at 1/20,000 and a competitor secondary (Right Image) at 1/50,000. Notice the increased background of the competitor product.Performed under reducing conditions.Observed band size: 42 kDa why is the actual band size different from the predicted?Exposure time: 5 secondsThis 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 ab8227 overnight at 4 C. Antibody binding was detected using ab205718 (Left Image) and a competitor secondary (Right Image), and visualised using ECL development solution ab133406. IHC image of beta tubulin staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4 C with ab6046 at 1/100 dilution. An HRP-conjugated secondary (Ab205718, 1/20000 dilution) was used to detect the primary for 1hr at room temperature. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre IHC image of Ki67 staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4 C with ab15580 at 1/1000 dilution. An HRP-conjugated secondary (Ab205718, 1/20000 dilution) was used to detect the primary for 1hr at room temperature. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre Cross-reactivity of the polyclonal secondary antibody ab182016 was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards at 1 g/ml(50 l/well) and incubatedovernight at 4 C, followed by a 5% BSA blocking step for 2h at RT. ab182016 was then added starting at 1 g/ml and gradually diluted 1/4 (50 l/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H L (HRP)(ab6885) was used at 1/10,000 dilution (50 l/well), followed by incubationfor 1h at RT.For the batch tested, ab182016 showed a cross-reactivity of 5-7% towards Human IgG and below 2% towards Mouse IgG, RatIgG and Chicken IgY.This data was developed using the unconjugated antibody (ab182016). Cross-reactivity of Goat anti-Rabbit IgG H L (ab182016)and Goat anti-Rabbit IgG H Lobtained from two different vendors was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards (Rabbit, Human, Mouse and Rat) at 1 g/ml (50 l/well) and incubatedovernight at 4 C, followed by a 5% BSA blocking step for 2h at RT. Secondary antibodies were then added starting at 1 g/ml and gradually diluted 1/4 (50 l/well), followed by incubationfor 2h. For the detection Donkey anti-Goat IgG H L (HRP)(ab6885) was used at 1/10,000 dilution (50 l/well), followed by incubationfor 1h atRT. This data is from a representative dilution.This data was developed using the unconjugated antibody (ab182016). To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on. Click here to view the general protocols Publishing research using ab205718? Please let us know so that we can cite the reference in this datasheet. ab205718 has been referenced in 1056 publications. Tanner AR et al. Impact of chorionic somatomammotropin RNA interference on uterine blood flow and placental glucose uptake in the absence of intrauterine growth restriction. Am J Physiol Regul Integr Comp Physiol 320:R138-R148 (2021).PubMed: 33146554 Chen Y et al. Increased ABCC2 expression predicts cisplatin resistance in non-small cell lung cancer. Cell Biochem Funct 39:277-286 (2021).PubMed: 32815556 Yu C et al. CircRNA TGFBR2/MiR-25-3p/TWIST1 axis regulates osteoblast differentiation of human aortic valve interstitial cells. J Bone Miner Metab 39:360-371 (2021).PubMed: 33070258 Cui Y et al. Upregulation of Fecal Epithelial Heparanase mRNA Is Associated with Increased Ulcerative Colitis Activity and Cancerization Risk. Dig Dis Sci 66:1488-1498 (2021).PubMed: 32445051 Zhang PF et al. Integrated analysis of phosphoproteome and ubiquitylome in epididymal sperm of buffalo (Bubalus bubalis). Mol Reprod Dev 88:15-33 (2021).PubMed: 33140506 View all Publications for this product Recombinant protein was incubated with a rabbit-raised primary antibody to generate a standard curve in order to quantify unknown samples. Detection was possible even on low amounts of protein and consistent between experiments. The first image was incubated with ab40763 and ab205718 (at 1/200)The second image is a negative control, with no primary antibody and ab205718 (at /200). Assay performed on whole cell lysate (mouse embryonic stem cells). Samples were mixed with 2X Laemli sample buffer and loaded on a 8% acrylamide gel together with Spectra™ Multicolor High Range Protein Ladder. Run performed in vertical tank at 100V for 1 hour followed by transfer in a wet system for 2 hours at 60V. The membrane was preblocked with 5% milk and then incubated with primary antibody (alpha-tubulin) overnight diluted in 5%milk. After washes the membrane was incubated with ab205718 1:2000 for 2 hours at RT. Bands were detected with ECL. First lane:ladder, second lane:wild type mouse ES cells (D3), third lane: E-cadherin knock-out mouse embryonic cell line.ab205718 worked for the chosen protein by WB. However, additional unspecific band detected. - 20ug of bovine liver lysates were loaded on to 4-15% gradient gel. - Blocking in 5% skim milk for an hour at room temperature.- 1:2000 AMPK alpha antibody (ab32047) were incubated over night @ 4C- 1:20000 secondary antibody (ab205718) used for an hour at room temperature. 10ug whole cell lysate (human ES cells).Blocking buffer - 5%BSA/1%OvAlb/PBS for 3 hours.Primary antibody - NR1D1(ab174309) 1:1000, 15 hours at 4C, expected size 67kDa.Secondary diluted in TBST (1:5000) for 3 hours at room temperature.ECL prime: 1 second exposure. Excellent specificity. Excellent sensitivity.WB conditions:- Cell lisates: 20 μg protein- non-reducing- Primary antibody: Rabbit anti-LaminB1, ab133741. Abcam. Dilution 1:5000- Secondary antibody: 1:6000- Developer: ECL Prime, Amersham, GE Helathcare Life Sciences, Product code RPN2232- Exposure time: 1 second, 2nd film out 4Please note: All products are FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES For licensing inquiries, please contact partnerships@abcam.com