Anti-Calmodulin 1/2/3 antibody [2D1] (ab2860)

Vielen Dank für Ihren Anruf und für Ihr Interesse an unseren Produkten.

Unter unseren anti-Calmodulin- Antikörpern ist ab2860 vermutlich der aussichtsreichste Kandidat: Das Immunogen (von Dictyostelium discoideum, SwissProtID P02599) weist eine Sequenzähnlichkeit von 83% gegen Calmodulin von Weizen (Triticum aestivum,P04464) auf. Daher ist eine Kreuzreaktivität von ab2860 sehr wahrscheinlich.

https://www.abcam.com/index.html?datasheet=2860 (or use the following: https://www.abcam.com/index.html?datasheet=2860).

ab2860 können wir für die Anwendungen ELISA, Western blot und Durchflusszytometrie mit diversen Spezies (Ratte, Huhn etc) garantieren. Unseres Wissens nach wurde der Antikörper aber bisher noch nicht in mit Weizen getestet. Falls Sie dies selbst testen möchten, kann ich Ihnen zur Zeit ein spezielles Angebot über einen 100%igen Abreview-Rabatt anbieten. Bei diesem Angebot bekommen Sie einen kostenlosen primären Antikörper, wenn Sie uns ein Abreview mit dem Testresultat zusenden. Um von diesem Angebot profitieren zu können, folgen Sie bitte diesen Schritten:

1.) Bestätigen Sie mir bitte, dass Sie ab2860 mit Weizen testen möchten.

2.) Bitte bestellen Sie erst nach Erhalt des Rabattcodes.

3.) Bestellen Sie den Antikörper wie üblich per Telefon, Email oder Fax

4.) Testen Sie den Antikörper mit Weizen.

5.) Teilen Sie uns das Ergebnis Ihres Tests durch ein Abreview mit und notieren Sie den Discount Code in dem Feld "additional notes". Unter der folgenden URL können Sie mehr über unser Abreview System erfahren: https://www.abcam.com/abreviews

6.) Der Rabattcode ist nach dem Abschicken des Abreviews aktiv, und Sie können einen anderen primären Antikörper bei uns bestellen (halten Sie bei der Bestellung bitte den Rabattcode und die Bestellnummer bereit). Bitte beachten Sie, dass der Rabattcode innerhalb von 4 Monaten nach Ausstellung eingelöst werden muss.

Der Rabattcode wird gültig unabhängig davon, ob Ihr Ergebnis positiv oder negativ ist.

Die Bedingungen zu unserem 100% Abreview Rabatt können Sie unter dem folgenden Link nachlesen: https://www.abcam.com/collaborationdiscount

Ich hoffe, diese Informationen helfen Ihnen weiter. Falls Sie einen Rabattcode erhalten möchten oder weitere Fragen haben, zögern Sie bitte nicht, sich wieder an mich zu wenden.

Thank you for your phone enquiry and questionaire. I'm sorry to hear you are having a problem with ab2860 (calmodulin Ab) in Western blotting. We have received other enquiries regarding this antibody and I would like to suggest the following modifications to your protocol: 1) Increase amount of protein loaded - try 50ug (others have even used 100ug) 2) Gel - increase gel percentage to 12-15%, this is better for smaller proteins; also, use PVDF membranes so smaller proteins won't run through 3) Blocking buffer - try straight milk in PBS and use straight PBS for washings (3X 5min) 4) Secondary Ab - does the product sheet suggest 1-15,000? Try 1:10,000. 5) Detection method - use ECL+, it's more sensitive than ECL. 6) Primary control - please include to know if weak signal is unspecific background staining. Please let me know if this helps, you can even e-mail me your blot! Do not hesitate to contact us for further advice.

Thank you for your enquiry. I browsed the reference, Cell Mot. Cytoskel., 18: 113-122, 1991. There are a few suggestions, taken from the article, that I can give. These researchers had difficulty with their proteins transferring through the membrane. They recommended PVDF as the retention of calmodulin was better. If using nitrocellulose, you can place two pieces to prevent the small proteins from passing through the membrane and into the buffer. The originator has tested this product in BL21 (E.coli) cell lysates. They loaded 40-50 ug of protein from the cell lysate, an 18% gel was used and transfer conditions were with a semi-dry at 1 ampere for 1 hour. Normal immunoblotting parameters followed. The same was done with rat brain except 100 ug of protein was loaded. The originator has not tested this product in Chlamydomonas reinhardtii, the cross-reactivity information comes from a third party. I have enclosed a specific protocol below that might be of assistance. Please let me know if I can be of further assistance. Western blot detection of Calmodulin 1. Run samples on SDS/PAGE discontinuous gel (4% stacking gel, 15% running gel). Soak for 15 minutes in KP buffer (25 mM KH2/K2HPO4 buffer, pH 7.0). 2. Pre-wet Immobilon PVDF transfer membrane (Millpore) briefly in absolute methanol, and rinse for 15 minutes in KP buffer. 3. Transfer in KP buffer at 20 V overnight at 4°C. 4. Incubate membrane for 45 minutes at room temperature in 0.2% (v/v) glutaraldehyde freshly prepared in KP buffer. 5. Rinse in KP buffer and then block in 2% (w/v) BSA in TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.2) overnight at 4°C or 1 hour at 37°C. 6. Rinse the membrane in TBS containing 0.05% (v/v) Tween-20, 3 times for 10 minutes each at room temperature. (WASH) 7. Incubate with Anti-Calmodulin Antibody diluted in TBS for 1 hour at 37°C. 8. WASH. 9. Incubate with HRP-conjugated secondary, diluted in 2% BSA (w/v), 10% normal goat serum (v/v), 0.05 % Tween-20 (v/v), in TBS, for 1 hour at 37°C. 10. WASH. 11. Incubate membrane in AEC for up to 30 minutes at room temp for color development. AEC Solutions (Store at 4°C) Solution A: 0.17 g 3-amino-9-ethylcarbazole (AEC) in 10 ml dimethylformamide. Solution B: 0.47 g succinic acid, 2.38 g sodium acetate, 0.08 g thimerosal in dH2O to yield 20 ml final volume. Solution C: 3% (v/v) hydrogen peroxide in dH2O. Before use, mix 0.1 ml A, 0.2 ml B, 0.1 ml C in 9.6 ml dH2O. *Procedural details taken from D. Hulen, et. al. Cell Motility and the Cytoskeleton, 18: 113-122, 1991. The procedure listed above is intended only as a guide. The concentration of primary antibody listed here is a one that has been reported by users to have worked with the above procedure. Various assay conditions require that the optimal working concentrations be determined by serial dilution. The results that you obtain may vary depending on experimental conditions and technique.

I'm very sorry to hear you are experiencing problems with ab2860. I would also expect the lysates in rat and E.coli samples to work with this antibody, however there may be several reasons for the lack of signal you are currently experiencing. The problem could be due to the incubation time which is only 1 hour. I would recommend to incubate overnight at 4C to give plenty of time for the antibody to bind to the membrane. Our recommendation of 1/500 was optimized on a blot of purified Dictyostelium calmodulin and your samples may need more concentrated antibody. Although you say that your samples should contain plenty of protein I would recommend to determine the exact protein concentration in your lysates and add 30-40ug lysate per well. I would also like to suggest using an ECl+ detection method as ECl is less sensitive, and to make sure the protease inhibitors in your samples are fresh. You mention having defrosted the antibody " was aliquoted, did not freeze/defreeze more than 2 times". We recommend to use a fresh aliquot every time as some antibodies do not tolerate re-freezing. You mention blocking in milk but do not mention how long for. In our experience milk can actually prevent binding of the antibody if applied too much; I would therefore recommend trying a one hour blocking stage and incubating the antibody in TBST only, and also to try 5%BSA 1hr. I hope you will find those recommendations helpful. If you still experience problems with those changes please do not hesitate to contact me again and I will arrange a refund if the antibody was purchased in the last 90days,

Thank you for those extra details about your protocol this helped me a lot as I looked deeper into the cross reactivity of this antibody with your samples: 293T and HeLa cells are from human origin and 3T3 cells are from mouse origin. This could be the reason for the lack of signal, the antibody has only been tested on Cow, Chicken, E. coli, Rat, Chlamydomonas reinhardtii and Dictyostelium discoideum, and though I couldn't find the immunogen sequence ( Dictyostelium discoideum calmodulin) I checked the homology of other proteins which cross reacted with ab2860, to have an idea of the homology between the species: for example the chicken protein and the human protein homology was 87%, which is high but not an exact match, therefore this can explain the lack of signal. We only offer a refund for antibodies not working in applications and species tested as detailed on the datasheet so I cannot offer you a refund, however I would recommend running a positive control of Dictyostelium cell lysate and to try an anti-calmodulin antibody which is known to cross react with most mammals (e.g. ab1288), I hope this will help you, please do not hesitate to contact me again if you need further assistance,

I'm sorry to hear you are having a problem with ab2860. We have not had a complaint about this antibody before. Thank you for taking the time to fill in our questionnaire, it helped me to understand your problem and it is useful to know that another antibody worked, pointing towards an incubation problem with ab2860 or a damaged antibody. Could you please clarify a few more details so I can understand even better your protocol: -what species are the samples -what blocking buffer was used and for how long -the dilution buffer used for ab2860 -the incubation time for ab2860 -have you tried a lower dilution? Our starting dilution of 1:500 is a recommendation and depending on the samples a final dilution may need to be used (eg 1:250-1:300)? Finally it would be good if I could find out your order details so I can look into possible delays in transport. I look forward to hearing from you to help you solve this matter, please be assured that should we find the antibody was at fault we will arrange a refund as requested, if you purchased the antibody in the last 90 days (I will need the order details to process the refund), My apologies for the delay,

Thank you for your enquiry and for your patience. Regarding the specific epitope mapping, we have not performed these studies. Both immunogens were: Calmodulin purified from Dictyostelium discoideum and both antibodies have been tested for application in ELISA. However, that is all the information that we have at this time. These two antibodies are only available in the unconjugated form. If you have any more questions, please do contact us again.