Anti-beta Defensin 1 antibody [M11-14b-D10] (ab14425)

Thank you for sending along the dot blots and additional information. We really appreciate the extra troubleshootingeffort.

Here are my thoughts:

1. In general it looks like the antibodies are able to detect Defensin in the chorioamniotic membrane samples.

2. It does not appear that the antibodies detected recombinant Defensin protein. You note the mass of recombinant protein spotted on the membranes, from 400ng - 0.78ng. Was the concentration of the Defensin proteinconfirmed by Bradford assay or OD280n measurements following reconstitution or are these number based on the expected concentrations after reconstitution?

If the concentrations are only theoretical, could you please check the concentrations to ensurethey are accurate?

Thanks again for your troubleshooting efforts. I'm sure we'll be able to resolve this situation soon.

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is are having problems with 8 of our products.  Special thanks for collecting the troubled customer's data/results in such an organized way. It may well be that the problem is either shipment/storage or protocol-related. Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.   1) Arrival dates: Could you please confirm the exact dates when the customer received these items: - ab14425, ab50048, ab66072, ab9872, ab19270, ab50059, ab14419, ab69499 2) Orders and shipment: - Could you provide me the Abcam Order Numbers for these products? - Were these items shipped in the same package or not? 3) Marker bands: - What MW markers (i.e. range of the proteins for MW) were used for WB? I am particularly interested in the markers at the lower weight range. 4) Image: - Could you please attach an image representing the samples and the loading control on the same gels? 5) Secondary antibodies: - Has the customer used the respective secondary antibodies successfully with other primary antibodies? Could you please check if the problem does not come from the 2ndaries?   Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Thank you for your enquiry. Human beta-1 defensin is expressed in kidney. Therefore, I would suggest you can use a human kidney whole cell lysate such as our peptide product ab30203. I have placed a link here to a reference I hope you may find useful from BMC Infectious Diseases; Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection (Jan Lehmann1 et al) http://www.biomedcentral.com/content/pdf/1471-2334-2-20.pdf Alternatively, you could use a purified beta-1 defensin peptide/protein. I hope this is helpful. Please do not hesitate to contact us again if you require more assistance.

Thank you for your enquiry. In testing this antibody, the plates were coated with linear and tri-cyclic ß-Defensins (1 µg/1ml) and a good signal was seen with an antibody-dilution of 0.1 µg/ml. Please find the protocol used in quality testing below. I hope this information helps, please do not hesitate to contact us if you need any more advice or information. We coated with linear and tri-cyclic ß-Defensins (1 µg/1ml) and we achieved a good signal having an antibody-dilution of 0,1 µg/ml. REAGENTS Coating buffer: 0.05 M Na2CO3 adjust pH to 9.6 with solid NaHCO3 Blocking buffer: 2% BSA in PBS Washbuffer: 1.37 Nacl, 26.8 mM KCl, 14.7mM KH2PO4, 81 mM Na2HPO4, 1 % TRITON-X-100, 0.02% THIMEROSAL First antibody: dilute in 2% BSA in PBS Second antibody: dilute in wash buffer Substrate : TMB-solution (BIOFX, USA) Stop solution: 0.4 M H2SO4 Microtiterplate NUNC Medisorb PROCEDURE • pipette 100 µl of peptide (1000 ng/ml in coating buffer) into the wells of a microtitre plate • incubate over night at 4°C • decant the contents of the plate and add 250 µl blocking buffer • incubate 1 hour at room temperature, shaking on a horizontal mixer • decant the content of the plate and wash the cavities 5 x with 250 µl of wash buffer • pipette 100 µl of antiserum (respective dilution in 2% BSA / PBS) • incubate at least 3 hrs at room temperature (or over night at 4°C), shaking on a horizontal mixer • decant the content of the plate and wash the cavities 5 x with 250 µl of wash buffer • pipette 100 µl of POX-labelled second antibody (e.g. if first antibody is rabbit: donkey anti-rabbit-POX can be used at 1: 10000 in wash buffer) • incubate 1 hr at room temperature, shaking on a horizontal mixer • decant the content of the plate and wash the cavities 5 x with 250 µl of wash buffer • pipette 100 µl TMB-solution • incubate for 15 - 30 minutes at room temperature, shaking slightly, until sufficient color differentiation has been observed • add 50 µl of stop solution and mix shortly • measure the extinction of the samples at 450 nm (reference wave length 620 nm)

Thank you for taking the time to submit this enquiry. The Ab that you have been utilising has not been specifically tested for use in dot blot Elisa but standard direct/indirect ELISA. I would be grateful if you could provide me with details relating to the protocol that you have utilised in order for me to look into this matter in more detail. My initial concern would be that a kit that has been specifically design for Western blot analysis is being utilised as a detection system. The “Western Breeze” procedure specifically mentions not allowing the membrane to dry out; however I believe that during dot blot ELISA the antigen is spotted onto air dried membrane (pre soaked in PBS) before being dried for a further hour. Do you have a standard protocol for dot blot ELISA within your lab, as I would be inclined to optimise a published dot blot protocol rather than utilising kits optimised for use in other applications. I look forward to hearing form you shortly.

Thank you for getting back to me with those details, it was very interesting. Unfortunately given that the source of the antisera have not been able to provide me with further details as to the synthesis of the immunising peptides it is difficult for me to comment on whether they have been raised against a correctly folded immunogen. If this was indeed the case then one might speculate that these antibodies only recognise the synthetic peptide and not the native form of the protein. However, checking back to past orders and this antibody is relatively popular and we have not received any complaints as such. I can but speculate that the absence of western or dot blot as applications on the datasheets is a reflection that these antibodies do not recognise their epitopes when immobilised on a membrane.

Thank you for your enquiry. Further to correspondence with the source of this antiserum I have been informed that the antiserum was raised against tricycle human ß-Defensin 2 antigen. Unfortunately they could not provide details of whether the antisera had been tested against the native protein or your protein standards. I have therefore not been able to find evidence that these sera have been tested against the native form of this protein. I would definitely be interested to know how these sera performed against the denatured forms of your standards.

We are sorry, but it is not known to us. It has not been tested.