Anti-VEGF antibody [5C3.F8] (ab3109)

Ich bedauere Ihnen mitteilen zu müssen, dass trotz Wechsel des Sekundärantikörpers (ab98691) leider noch immer keine positiven Signale vorhanden sind. Ich habe das Protokoll ansonsten beibehalten. Hätten Sie vielleicht noch weitere Vorschläge, die zu einem positiven Resultat führen könnten? Vielen Dank für Ihre Mühe.  

ANSWER:

Vielen Dank für Ihre Antwort und für diese weiteren Informationen.

Es tut mir sehr leid zu hören, dass Sie immer noch keine positiven Signale erhalten haben.

Zur Optimierung des Protokolls fallen mir leider nur zwei Punkte ein:

1) Die Inkubation mit dem Primärantikörper bei 4°C (über Nacht) führt in der Regel zu sehr spezifischen und stärkeren Ergebnissen als eine Inkubation bei höhererTemperatur. Falls Sie dies nicht schon probiert hatten, könnte es durchaus zu einer Verbesserung des Signals kommen.

2) Die Antigen-Demaskierung mit Citrat-Puffer bei pH 6.0 ist eine Methode, die oft zum gewünschten Ergebnis führt. Für manche Antigene kann sich unter Umständen jedoch eine andere Retrievalmethode besser eignen. Dies muss für jedes Target individuell bestimmt werden. Vielleicht erzielen Sie mit Tris/EDTA-Puffer pH9.0 positive Signale? (Details siehe Anhang).

Ich weiß, dass Sie schon viel Zeit in Ihre Experimente investiert haben und es ist enttäuschend, dass Sie bisher noch kein positives Ergebnis haben. Sollten die Vorschläge nicht zum Erfolg führen, werde ich Ihnen daher gerne umgehend einen Ersatz bzw. eine Gutschrift schicken.

Vielen Dank für Ihre Kooperation und Hilfsbereitschaft. Ich freue mich, bald wieder von Ihnen zu hören.

Hi, Thanks for your excellent products. I have bought two primary antibodies from your company (ab64569 and ab3109). Now I want to detect changes in Endostatin and VEGF in skeletal muscle and tumor tissue but I don’t know characteristics of the lysis buffer for these antibodies. I ask you please suggest an appropriate lysis buffer for these antibodies. Yours sincerely,  

ANSWER:

Thank you for contacting us. I presume you want to perform a WB assay with both antibodies. There isn’t a specific lysis buffer indicated to lysate the samples, but in both cases denaturing/reducing conditions are required. In that case I would use an appropriate lysis buffer, such as RIPA, and I would strongly recommend using protease inhibitors in the buffer (a list can be found in our protocols section). It is also important to boil the samples in order to denature the proteins. I would recommend you to have a look through our WB protocols, where you could find detailed information about the sample preparation as well as buffer preparation and protocol steps: http://www.abcam.com/index.html?pageconfig=resource&rid=13045 I hope this information is useful. Please do not hesitate to contact us if you need further details or information.

LOT NUMBER GR53220 ORDER NUMBER 975372 DESCRIPTION OF THE PROBLEM No staining SAMPLE mouse: lung, pancreas, CT26-Tumor, LPS-liver PRIMARY ANTIBODY 1:25 and 1:50 in PBS incubation overnight at roomtemperature DETECTION METHOD Steaptavidin-POD, 1:50, 10 min at RT POSITIVE AND NEGATIVE CONTROLS USED negative control: sections without primary antibody ANTIBODY STORAGE CONDITIONS -20°C FIXATION OF SAMPLE 4% Formalin ANTIGEN RETRIEVAL Citrat-buffer pH 6.0, microwave 600 Watt, 10 min PERMEABILIZATION STEP none BLOCKING CONDITIONS 3% H2O2 in Methanol for 10 min 3% Goatserum in PBS 30 min Endogenous Avidin + Biotin Blocking System (ab3387) SECONDARY ANTIBODY goat-anti-mouse biotin (ab98696, 1:100, 45 min at RT HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? dilution of primary antibody ADDITIONAL NOTES we used pancreas as postitive control because of the pictures in the datasheet

ANSWER:

Vielen Dank für Ihre Anfrage und dafür, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen.

Es tut mir leid, dass Sie Probleme mit unserem VEGF-Antikörper ab3109 hatten. Ich weiß, dass Sie schon viel Zeit in Ihre Experimente investiert haben und es ist enttäuschend, dass Sie bisher noch kein positives Ergebnis haben.

Ich kann Sie darin bestätigen, dass die Pankreasschnitte auf jeden Fall positive Ergebnisse zeigen sollten. Laut Literatur eignet sich Lungengewebe auch als gute Positivkontrolle; Sie haben sich daher auf jeden Fall die richtigen Kontrollen ausgesucht.

Bei der Durchsicht Ihres Protokolls ist mir aufgefallen, dass Sie als Sekundärantikörper ab98696 verwenden: Dies ist ein Antikörper gegen Maus-IgG2a, der noch weiteren Aufreinigungsschritten unterzogen wurde (pre-adsorbed), um eventuell vorhandene Kreuzreaktivitäten zu minimieren. Daher ist es sehr unwahrscheinlich, dass ab98696 den anti-VEGF- Antikörper ab3109 bindet, da dieser Primärantikörper vom IgG1-Isotyp (mit der kappa light chain) ist. Ich möchte Ihnen daher empfehlen, einen anderen Sekundärantikörper zu verwenden (zum Beispiel ab98691 Click here (or use the following: http://www.abcam.com/index.html?datasheet=98691).).

Bei einer Inkubation ueber Nacht würden wir zwar eine Inkubationstemperatur von 4ºC empfehlen, da dies zu einem spezifischeren Signal führt. Abgesehen davon sieht Ihr Protokoll aber sehr gut aus und sollte positive Ergebnisse mit ab3109 und Ihren Proben bringen.

Bitte lassen Sie mich wissen, ob ich Ihnen helfen konnte und zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Thanks for your reply and suggestions. And I have forward them to the customer.

1. With regard to M-PER, it is a good universal reagent to extract protein and the customer doesn't think it has problem. In fact he did many western blot with this reagent. 2. Protein wasn't degraded because GAPDH control result is ok, and some other antigen were also detected with respective antibodies. 3. With regard to reducing condition, the customer applied DTT to PIERCE commercial non-reducing loading buffer. And he told me that he always uses reducing condition. 4. As for positive control, although recombinant VEGF was not used, his sample cell of gastric cancer cell was highly express VEGF and could be used as a control.

Based on them, would you please issue a credit note for this customer so that he could buy some other antibody? The PO number is 20060717Abcam, and it's ordered in July 18 2006 or so. Thank you very much.

ANSWER:

Based on this additional information I would be happy to offer a credit note for Jingmei for 1 vial ab3109.

Thank you for offering a refund to the customer,

This is China Jingmei Biotech Ltd. A customer just purchased ab3109 and tried western blotting. Unfortunately he got no bands. Here is the questionnaire:

1. Order details: . Batch number: Lot: 155784 . Abcam order or Purchase order number: ab 3109-500 . Antibody storage conditions (temperature/reconstitution etc) Stored in 4 degree refrigerator

2. Please describe the problem (high background, wrong band size, more bands, no band etc). No band, while the GAPDH in the same membrane is clear. (see attachment photos)

3. On what material are you testing the antibody in WB? . Species: human gastric cancer cell lines . Cell extract or Nuclear extract: cell extract . Purified protein or Recombinant protein: purified protein

3. The lysate . How much protein was loaded: 100 ug . What lysis buffer was used: M-PER mammalian protein extraction reagent (PIERCE) . What protease inhibitors were used: PIC+PMSF . What loading buffer was used: Non-reducing lane marker sample buffer (PIERCE) . Did you heat the samples: temperature and time: 100 degree heated for 5 min

4. Electrophoresis/Gel conditions/ Transfer conditions . Reducing or non reducing gel: reducing gel . Gel percentage : separating gel:10%, stocking gel 4% . Transfer conditions: we tried twice : semi-dry transfer (15v for 1hr) and 5. Blocking conditions (we didn't block) . Buffer: . Blocking agent: milk, BSA, serum, what percentage: . Incubation time: . Incubation temperature:

6. Primary Antibody . Specification (in which species was it raised against): Mouse derived . At what dilution(s) have you tested this antibody: 2 ug/ml in TBS . What dilution buffer was used: TBS . Incubation time: 12hr . Incubation temperature: 4 degree . What washing steps were done: TTBS

7. Secondary Antibody . Specification (in which species was it raised against)? : anti-mouse Ig G . At what dilution(s) have you tested this antibody: 1:1000 . Incubation time: 2hr in room temperature . Wash steps: TTBS for 3 times, 10min/time . Do you know whether the problems you are experiencing come from the secondary? No 8. Detection method ECl, ECl+, other detection method: ECL 9. Background bands (we have no bands) . Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a "No primary" control): . Is the blocking step sufficient? . Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) . At what size are the bands migrating? Could they be degradation products of your target? . Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

11. Did you apply positive and negative controls along with the samples? Please specify.: This is our first time to detect the VEGF expression by WB. So we have no positive control samples. But it's reported that gastric cancer cell express it.

10. Optimization attempts . How many times have you tried the Western? 3 times . Do you obtain the same results every time e.g. are background bands always in the same place? Yes, we do. . What steps have you altered? We progressively increased the concentration of the primary antibody, but still has no band.

Dear Sir or Madam, based on his protocol and result, would you please issue a credit note for this customer as he applied? The PO number is 20060717Abcam, and it's ordered in July 18 2006 or so. Thank you very much.

ANSWER:

Thank you for providing your customer's protocol. I can understand how frustrated he must feel not having a good signal. I think the customer's protocol is very good in general and have only a few (but important) alterations to recommend. We do not have experience of the lysis buffer M-PER mammalian protein extraction reagent but it seems it should work (it may be worth trying a Tris or NP40 buffer in case the M-PER buffer is not suitable to extract VEGF), and I would strongly recommend to use more protease inhibitors in the buffer (a list can be found in our protocols section, or the customer can purchase a cocktail from Roche). Secondly, the customer should use a reducing loading buffer as non reducing conditions do not work for ab3109, so the customer must use a buffer containing reducing agents, then boil the samples in this buffer.

The main point is, as the customer says, that he has no positive control, and that his samples may be expressing low levels of VEGF165, making the detection very hard. When ab3109 was tested in western blotting it was tested against VEGF165 recombinant protein, so I would recommend to run this along the customer's samples.