Anti-BMP5 antibody (ab88064)

I am trying to do a direct ELISA but no matter what conditons I try, I am not seeing any signal using this antibody. I have tried different ab concentrations and blocking conditions.

ANSWER:

I am sorry that you were having problems with ab88064, mouse BMP5. I have processed the free of charge replacement for ab93852, rabbit polyclonal to BMP5. Unfortunately, it now saying that it is  out of stock and there is a current lead time of 1-2 weeks for this antibody to come back into stock. If this will not work for you, please let me know. Your new order # is 968309. Please let me know if there is anything else I can do for you.

I am trying to do a direct ELISA but no matter what conditons I try, I am not seeing any signal using this antibody. I have tried different ab concentrations and blocking conditions.

ANSWER:

 Thank you for contacting Abcam about ab88064, the anti-BMP5 antibody.   They have confirmed that the anti-BMP5 antibody has been tested in ELISA using recombinant BMP5 protein and they have provided the protocol that they used (see below). Please let me know if there is anything else that I can do to help, or if you would like to test out the other BMP5 antibody (ab93852) that we talked about on the phone. ELISA Protocol 1. Coat antigen (200ng/well) onto the wells in a 96 well mictrotiter plate 2. Block unbound sites with 5% skim milk in PBST 3. Apply hybridoma culture supernatant/ ascites/ purified Ig as primary antibody. Incubate the plate at room temperature for two hours. 4. Wash 4 times with PBST. 5. Apply HRP conjugated secondary antibody, and incubate the plate at room temperature for one hour. 6. Wash 8 times with PBST. 7. Apply 100ul OPD in citric acid buffer, and incubate at room temperature for 20minutes. Read the plate in ELISA reader at 450 nm. ※ Between each step, plates were adequately washed using PBST. ※ Secondary antibody dilution, 1:1000 Primary Antibody Dilution factor/ Concentration Diluents Poly sera 1500X Cultured Supernatant 1X Ascites 1000X Purified Ig 1ug/ml 5% skim milk in PBST Material: PBST, 0.2% Tween 20 Citric acid                

I am trying to do a direct ELISA but no matter what conditons I try, I am not seeing any signal using this antibody. I have tried different ab concentrations and blocking conditions.

ANSWER:

Thank you for contacting Abcam about ab88064.  Please find the protocol that was used the sandwich ELISA below. I have contacted the lab to see if the antibosy has been tested in a direct ELISA. If there is anything else I can do in the mean time please let me know. SECTION 1 – Reagents 1.1 Coating Buffer PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5mM KH2PO4) 1.2 Blocking Solution 5% Skim Milk in PBST (0.05% Tween-20) 1.3 Diluent 2% Skim MILK in PBST (0.02% Tween-20) 1.4 Citrate Buffer 3.65 g citric acid, 4.76 g Na2HPO4 in 500ml ddH2O 1.5 Rabbit anti-GST antibody Abnova’s rabbit anti-GST antibody 1.6 HRP conjugated goat anti-rabbit IgG (H+L) PIERCE Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated SECTION 2 - Assay Protocol Note: The following protocol is a guideline, user need to determine their optimal experimental condition for best performance. 2.1 Apply capture antibody by adding antigen-specific antibody to appropriate wells (1μg/well).The antibody concentration should be 10 μg/mL in coating buffer, the volume should be 100 μL/well. The capture antibody should be purified antibody. ※ 2.2 Incubate the plate at 4°C overnight. 2.3 Add 250 μL of blocking solution each well. 2.4 Incubate the plate at room temperature for 1 hour. 2.5 Empty the plate and wash the plate with PBST (0.05% Tween-20) once. 2.6 If the analytes is: a 、 Recombinant protein - dilute it to 100 ng, 30 ng, 10 ng, 3 ng, 1 ng, 0.3 ng, 0.1 ng, and 0.03 ng/mL in diluents. b 、 Transfected lysate - dilute it at serial dilution folds to determine adequate dilution folds which fit the standard curve. Abnova usually uses 3X, 10X, 30X, 100X, 300X, 1000X, 3000X at this step. 2.7 Add analytes to appropriate wells. 2.8 Incubate the plate at room temperature for 2 hours. 2.9 Empty and then wash the plate three times with PBST (0.05% Tween-20).   2.10 Apply detection antibody by adding tag-specific rabbit anti-GST antibodies to appropriate wells (2.5 μg/mL, 100 μL/well). 2.11 Incubate the microtiter plate at room temperature for 2 hours. 2.12 Empty and then wash the plate three times with PBST (0.05% Tween-20). 2.13 Apply secondary antibody by adding HRP conjugated goat anti-rabbit IgG (H+L) to appropriate wells. 2.14 Incubate the microtiter plate at room temperature for 1 hour. 2.15 Wash the plate 5 times with PBST (0.05% Tween-20). 2.16 Apply the substrate by adding 150 μL of substrate (OPD, 400 μg/mL, 0.03% H2O2, citrate buffer) 2.17 Incubate at room temperature for 30 minutes. 2.18 Read absorbance at 450 nm.