Question (63888) | Anti-Survivin antibody [60.11] (ab93274)

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Question

1) Abcam product code ab 93274 lot GR88156-1
2) Abcam order reference number or product batch number 1160799
3) Description of the problem I did not obtain any staining
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysates
Lysis buffer Laemmli Buffer
Protease inhibitors: no
Phosphatase inhibitors no
Reducing agent DTT
Boiling for ≥5 min? yes
Protein loaded ug/lane or cells/lane 400.000 cell /line or 10- 50 or 90µg/lane
Positive control The hematopoietic cell lines are positive for survivin (for example K562 or LAMA84). I detected survivin on 5µg of protein with the antibody code ab7170 clone 60.11
Negative control
5) Percentage of gel 12%
Type of membrane nitrocellulose
Protein transfer verified Ponceau Red stain
Blocking agent and concentration 5% nonfat dry milk in TBST 0.2%
Blocking time 1 hour
Blocking temperature room temperature
6) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1:200, 1:500 or 1:1000
Diluent buffer 5% nonfat dry milk in TBST 0.2%
Incubation time O.N.
Incubation temperature: 4°C
7) Secondary antibody:
Species: from sheep
Reacts against: Anti-mouse IgG
Concentration or dilution 1:1000
Diluent buffer 5% nonfat dry milk in TBST 0.2%
Incubation time 1 hour
Incubation temperature: room temperature
Fluorochrome or enzyme conjugate: Horseradish peroxidase
8) Washing after primary and secondary antibodies:
Buffer TBST 0.2%
Number of washes 3 times for 5 min each
9)Detection method LiteAblot system from Euroclone. I exposed the membrane for 15 minutes
10) How many times have you run this staining? 3 times
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody? The concentration of antibody and the µg of proteins
Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.
N.B. I think that the problem is the purity of the antibody.
The purity of the old mouse monoclonal 60.11 to survivin (ab7170) is ascites, instead the purity of the anti-survivin clone 60.11 is protein G purified.
I would like to buy the old antibody.
Is it possible?
Thank you in advance for your interest
Elena Tirrò
First lane marker
Second lane K562 whole lysate 10µg
Third lane K562 whole lysate 50µg
Fourth lane K562 whole lysate 90µg
Fifth lane LAMA84 whole lysate 50µg
Sixth lane LAMA84 whole lysate 90µg

Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

The ab7170 antibody is out of stock which is why it was unpublished. I am sorry we will not be able to provide this product. The product ab93274 is indeed a purified antibody which however will not be affecting the specificity of this antibody.

I have few following further questions about the protocol;

- The image is totally clean so could I double check? have you used 400 cells/ well or 50- 90ug protein/ lane?
- Did you observe same clean membrane after trying 3 times?
- Is the secondary antibody fine? Have you tried "loading control" with the same lysates?
- Is it true no protease inhibitors were added? Could you try after adding a PI cocktail?

Finally would you like to try a different lot of this product.

I will look forward to hearing from you soon.

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