Anti-Calmodulin antibody [2D1] (ab2860)

Hallo,
ich benötige einen Calmodulin Antikörper, der gegen Calmodulin aus Wheat Germ gerichtet ist. Haben Sie solch einen Antikörper im Programm bzw. welchen könnte ich am besten nehmen? Können Sie mir dazu weitere Informationen geben?
Vielen Dank!

ANSWER:

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.

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

BATCH NUMBER 167969 ORDER NUMBER 1638544

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Bovine rod outer segments

PRIMARY ANTIBODY ab2860-200 1:500 dilution in blocking buffer ON incubation 3x10min. washes in blocking buffer

DETECTION METHOD ECL

ANTIBODY STORAGE CONDITIONS Aliquoted and stored at -20C

SAMPLE PREPARATION Bovine rod outer segments were isolated from retinas by gradient cetrifugation. The retinas were homogenized in 30% sucrose, 65mM NaCl, 2 mM MgCl2, 5 mM Hepes pH 7.4 and separated on a discontinuous sucrose gradient in 10 mM Hepes and 1 mM MgCl2. Isolated rod outer segments were resuspended in 10 mm Hepes pH 7.4 and solubilized with the addition of 1% NP-40.

AMOUNT OF PROTEIN LOADED 30 micrograms, 15 micrograms, 3 micrograms, 1.5 micrograms

ELECTROPHORESIS/GEL CONDITIONS reducing (5% BME) 10 % Bis-Tris Invitrogen gel; MES buffer

TRANSFER AND BLOCKING CONDITIONS Transfer buffer (Invitrogen)/ 1 hour Blocking buffer: 5% non-fat milk in PBS pH 7.4 with 0.1% NP-40

SECONDARY ANTIBODY anti-mouse IgG horse radish peroxidase conjugated (Pierce)1:15,000 dilution/ 2 hours incubation 3x10 min. washes in blocking buffer blot store in PBS

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

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.

Customer is using this antibody in Western blot on Chlamydomonas reinhardtii, and is seeing higher molecular weight bands, but no bands around 17kDa where expected.

Sample: 20ul of lysate, protein concentration not known Block: 5% milk in TBST, RT 1h, also tried 2h 1: 1:500 in TBST with milk and without milk (no difference) O/N 4C 2: tried an AP and an HRP secondary. ECL+ was way too sensitive, black blot.

ANSWER:

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.

BATCH NUMBER 130172 ORDER NUMBER 119538

DESCRIPTION OF THE PROBLEM No signal at all

SAMPLE Tried 4 different species/cell extracts: CHO, HEK293, PC12 and DH5alpha bacteria (E.Coli).

PRIMARY ANTIBODY Mouse mono to Calmodulin (ab2860-200) 1:500 as recommended, incubation at least 1hour, wash 4 times in PBS-0.5%tween for 10 min each with gentle rocking (also tried 1:1000)

DETECTION METHOD ECL from Amersham

POSITIVE AND NEGATIVE CONTROLS USED none

ANTIBODY STORAGE CONDITIONS -20C, aliquoted, did not freeze/defreeze more than 2 times

SAMPLE PREPARATION PC12(rat): lysed 2 confluent 10 cm plates in 500ul RIPA (1%NP40,0.1%deoxycholic acid, PBS pH 7.4, 1tablet prot.inh.) CHO and HEK293:lysed 2 confluent plates in 500 ul RIPA (0.5%NP40) DH5alpha: grow in 5 ml 2YT, spin, remove supt, lysed in 500ul GST lysis buffer(0.5%NP40, 1mMEDTA, 1 tablet prot.inh (Roche),PBS) NEVER heated any of the lysates above 4C.

AMOUNT OF PROTEIN LOADED PC12 and DH5alpha: used 6X gel loading buffer,so I could load 40ul of lysate CHO and HEK293: loaded 25ul of lysate Did not measure the protein concentration, but this is what i load for other experiments and the other antibodies I use work fine.

ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE 10%

TRANSFER AND BLOCKING CONDITIONS Transfer ON at 35 volts in Transfer buffer (57.7 g Glycine, 12 g Tris, 800 ml MeOH in 4L water) Blocking: 5% skim milk

SECONDARY ANTIBODY HRP anti mouse, 1:5000, [ a competitor], at leas 1 hr in 5%milk-PBS-tween, wash 4 times in PBS-tween

HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ADDITIONAL NOTES I have no signal at all even if I expose the film ON, I think that this batch of antibody might be bad. Maybe it does not react with human or mouse, but I don't see why it would not react with RAT or E.Coli. I don't know if it would work better at dilutions such as 1:200, but I think that this would be ridiculous and I would not want such a poor antibody anyway. If possible, we would like to be refunded, because we have wasted enough of time waiting for the antibody (3 weeks) and then testing it. We would like to simply buy it from another company.

ANSWER:

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,

Follow up to previous enquiry: Thank you for your reply. I also feel sorry for the problem of this anti-calmodulin antibody (ab2860).

I have tried nuclear lysates prepared from 293T cell, Hela cell and 3T3 cell, but the endogenous calmodlin from none of them could be detected by ab2860 on Western Blots. The blocking buffer I used was TBST-milk(5%), 30mins incubation. The dilution buffer used for ab2860 was also TBST-milk (5%), and with overnight incubation. The dilution I used actually was 1:400. I won't be satisfied if it works in even lower dilution.

Hope these details will help you somehow.

I ordered this antibody on Jun 22nd. If it's possible, I would still prefer to get the refunding. The Purchase order No is 40206676 /H/A03/A030 and please tell what else would you like to know?

Thank you anyway for your nice service! Hope your company will solve this problem soon!!

ANSWER:

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,