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oM of our costumers is having problems with:
ABCAM Goat polyclonal anti-mouse IgA alpha chain - DyLight 650
P.O. # 0076/12 (March 2012)
She is using ICC/ IF technique and says that the antibody isn't giving the desired signal. It's negative or too weak on her cell preparations (see attached images).
Protocol tested with ab97014:
1- For cell culture were used 24 well plates with one 2% gelatin covered circular glass coverslip inside each well.
2- Cells from CACO-2 line (human epithelial colorectal adenocarcinoma cells) were cultivated for 16 Hours in 1 mL of DMEM with 10% FBS Cultire Medium per well.
At that stage, approximated 10000 cells were in the culture. This number of cells is not confluent, so allow to take pictures of isolated cell groups (2 to 5 cells only).
3- After 16 Hours, cells were washed with PBS (1X) and fixed with 3% Formalin for 10 minutes.
4- Washes with PBS (3X).
5- Cell permeabilization: 0.05% Saponin/PBS for 10 minutes.
6- Washes with PBS (3X).
7- Protein block: 1% FBS in PBS for 10 minutes.
8- Primary antibody: Monoclonal mouse anti-ADRP (Fitzgerald) diluted 1:75 in 0.05% Saponin/PBS for 1 hour, at room temperature.
9- Washes with PBS (3X).
10- Secundary antibody: ab97014 diluted 1:50 in 0.05% Saponin/PBS for 45 minutes, at room temperature.
11- Washes with PBS (3X).
12- Bodipy (488 nm) incubation for 45 minutes, at room temperature.
13- Washes with PBS (3X).
14- DAPI incubation for 5 minutes, at room temperature.
15- Wash and mounting of the coverslips on glass slides.
16- Slides were analyzed on Confocal Microscope (Fluoview FV10i Olympus)
1- As a positive reaction control the same conditions exposed above were also used with secundary antibody anti-mouse Rhodamine from KPL manufacturer and the reaction signal was OK.
2- Some variations of the protocol mentioned above have already been tested with ab97014 all without success:
- Increase permeabilization time using 0.05% Saponin (Protocol - Step 5): 30 minutes
- Change permeabilization reagent (Protocol - Step 10): 0.2% Triton/ PBS
- Change permeabilization reagent (Protocol - Steps 5 and 10): 0.2% Triton/ PBS
Even with these protocol changes, the reaction signal was still negative or too weak.
What do you think about the case?
1-) Could this be a problem with the reagent vial (ab97014 - Batch GR76514-1)?
2-) If you think the antibody is OK, can you give us any clue to improve protocol?
I'm forward to hearing from you.
Thanks for your attention.
Asked on May 29 2012
rrding ab97014 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this secondary antibody.
As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
1) Primary antibody - Could you specify the followings:
- Isotype - Is it compatible with the secondary antibody?
- Has it been tested for ICC/IF on human cells?
Has the customer applied any other permeabilzing agents such as acetone or TBS-T?
3) Positive control:
Has any positive control be applied parallel with the samples?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.
I look forward to hearing from you soon.
Answered on May 29 2012