Anti-Survivin 2B antibody (ab3729)

Gerne bestätige ich Ihnen, dass ich die Antikörper für die IHC-P austesten möchte.

ANSWER:

DISCOUNT CODES: ab3729: xxxxx ab3731: xxxxx Ablaufdatum (jeweils): xxxx2012

Es freut mich zu hören, dass Sie unser Angebot unseren Antikörper ab3729 und ab3731 in der Immunhistochemie an Paraffinschnitten (IHC-P) zu testen, annehmen möchten. Wie am Telefon besprochen, können Sie den Gutscheincode jeweils zum Kauf eines weiteren Antikörpers einsetzen, sobald Sie uns ein Abreview für diesen Antikörper eingereicht haben. Die Discountcodes haben obige Nummern und können zum Kauf weiterer Produkte eingesetzt werden. Der Code wird jeweils gültig, sobald Sie uns ein Abreview für diesen Antikörper über den Test in der IHC-P eingereicht haben. Bitte geben Sie diesen Code auch im Abschnitt “Additional Notes” des Abreviews mit an, so dass wir wissen, dass sich dieses Abreview auf die Gutscheinaktion bezieht. Der Code wird dadurch aktiviert. Bitte kontaktieren Sie bei der nächsten Bestellung unsere Kundendienst mit dem Code und der ursprünglichen Bestellnummer. Bitte zögern Sie nicht, sich wieder an uns zu wenden, falls Sie weitere Fragen haben. Wir freuen uns auf Ihr Abreview, egal ob Ihre Ergebnisse positiv oder negativ sind und wünschen Ihnen viel Glück für Ihre Forschung. Die Bedingungen dieses Angebotes befinden sich unter dem folgenden Link: www.abcam.com/collaborationdiscount  

I have another question. I have been looking through the Survivin 2B amino acid sequence. For Survivin 2B [ab3729],the synthetic peptide that was used as the immunogen was derived from residues 50-150 of human Survivin 2B. While some of the amino acids are specific to Survivin 2B, there are also amino acids conserved with Survivin and Survivin-Ex3. Is it possible, then, that this antibody will also detect survivin and survivin DEx3 ? Thanks again for your help,

ANSWER:

Thank you for your enquiry.

This antibody has been tested in Western blotting against equal loadings of purified wild type survivin, 2B and DEx3. When used at 1/500, it detects a single clean band of the appropriate size for 2B, but no band is detected with wild type survivin or DEx3.

Don't hesitate to contact us if you have additional questions.

We are currently using ab3729 for Western blotting. Does abcam sell a positive control for this antibody ? Also, would you please recommend a secondary that you have tried for your in house testing of this primary ?

ANSWER:

Thank you for your enquiry and your patience.

We do not sell a positive control for this antibody. We have not succeeded in detecting endogenous Survivin 2B using this antibody (Survivin 2B is expressed at very low levels). This antibody has been used successfully, however, to detect overexpressed Survivin 2B in whole cell extracts. I would recommend ab6721 as the secondary to be used with this primary.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

these is the additional information:

>1) Batch number >ab3729: 57185 >ab2050: 38860 > >2)Purchase order number >BIOZOL purchase > >3)Antibody storage conditions (temperature/reconstitution etc) >-20C > >4)Description of the problem (high background, wrong band size, more bands, >no band etc.) > no bands/non specific bands > > >5)Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant >protein etc.) >soft tissue sarcoma (human) cell lines > >6)Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) >Buffer: Bradford- Buffer/Protease inhibitors: Protease Inhibitor Cocktail (Sigma) 1:100 / Heating sample 70°C for 10 min > loading Buffer: NUPAGE LDS-Sample NP 0007 ( Invitrogen)

>7)Amount of protein loaded >50ug > >8) Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the >gel etc.) >Reducing gel (NP0009 Invitrogen), 12 % Bis-Tris-Gel (NP0322 Box Invitrogen) 120 V and 120 mA for 1,5 hours > >9)Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) >5% milk 3hrs > >10)Primary Antibody (Diluent/Dilution/Incubation time, Wash step) >ab3729 1:500 >ab2050 1:500 >overnight incubation 4C >yes >is the antibody diluted in milk? in buffer with tween? >yes 1% milk with TBS-T >11)Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation >time, Wash step) >DaKo 1:2000 > >does the antibody work with other primaries know to give a good signal? yes >Have you tested it with no primary? no, but that is not the reason, why we found to many bands ( because the band are different from antibody to antibody, although we used the same secondary antibody >Is it diluted in milk? yes 1% > >12)Detection method (ECL, ECLPlus etc.) >ECL > >13)Positive and negative controls used (please specify) >we don't have any positiv control for ab3729 or ab3731 (Do you have?) > >14)How many times have you tried the Western? > > >more than 5 times (every time we found more bands and non specific bands) > >15)Have you run a "No Primary" control? >No > >16)What steps have you altered? >we used fresh buffers, new blots other samples >

ANSWER:

I'm sorry to hear you are having a problem with ab3729 and ab2050.

I would like to suggest the following modifications to your protocol:

1) use a higher percentage gel to separate the protein adequately, this will enable better visualisation of the bands

2)following lysis of the tissue (always at 4C) mix the sample with loading buffer containing reducing and denaturing agents. The buffer you currently use (NP 10007) does not seem to contain those.

3) block the membrane in BSA 5% in TBST for 1 hr, then rinse quickly with TBST and add your primary antibody diluted in TBST (NO milk)

4) Please note the following comments are on our datasheet:

Ab3729 This antibody has been tested in Western blotting against equal loadings of purified wild type survivin, 2B and DEx3. When used at 1/500, it detects a single clean band of the appropriate size for 2B, but no band is detected with wild type survivin or DEx3. We have not succeeded in detecting endogenous Survivin 2B using this antibody (Survivin 2B is expressed at very low levels). This antibody has been used successfully however, to detect overexpressed Survivin 2B in whole cell extracts.

This may explain why you cannot detect survivin 2B at high signals.

For ab2050, please use Molt4 whole cell lysate as positive control. As I mention in my previous e-mail to your colleague, there are numerous bands with the other antibodies the researcher is using, suggesting the samples may be damaged. Protease inhibitors must always be added fresh to the lysis buffer and damage of one inhibitor in the cocktal may explain the problem.

I hope this information helps, please do not hesitate to contact us for further advice,

we have been working with the new Survivin-2B and Survivin Delta Exon-3 antibodies. We have had good success with the Exon-3 antibody and seeing bands on Western analysis in both transfected overexpressed cell lines and endogenous tissues. The 2B antibody, however, only shows bands on transfected overexpressed cell lines. Iwas wondering if you have had any success on any type of endogenoustissue using the 2B antibody? Of course, it may just not be translated in the tissues we have been testing, but I was curious if you have had any success with any type of cells/tissues? I would be curious to know what cell lines/tissues these are, if possible? Thanks for your time.

ANSWER:

Thank you very much for your email. Can you confirm that you are working with ab3729? Ab3729 has been tested in Western blotting against equal loadings of wild type survivin, 2B and DEx3. When used at 1/500, it detects a single clean band of the appropriate size for 2B, but no band is detected with wild type survivin or DEx3. We have not succeeded in detecting endogenous Survivin 2B using this antibody. This is all the information that we have at this time. Please let me know if you have any further questions.