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Problem (High background, No signal or week signal, Non specific staining):
A very week signal
Antibody storage conditions (temperature/reconstitution etc):
Description of the problem (high background, low signal, no signal etc.):
The signal was very low and when stain together with other flurofors it was difficult to estimate the results
Sample (Species/Cell type/Cell line etc.):
Human tumor cells growing in SCID mouse
Sample preparation for single cell preparation (Buffer etc.):
The tumor was cut to small piece and was squashed to get single cells in PBS Buffer
Number of cells used:
Permeabilization, fixation step:
Blocking conditions (Buffer/time period, Blocking agent etc.):
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):
Ab31891 was used in different concentration from 1 to 20ug as recommended there was no difference in the signal. 45 min incubation washed with PBS+5% FCS
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):
Positive and negative controls used (please specify):
Positive control for the Human cels of the tumor we used APC a CD326, Negative control was Fl Human IgG
Optimization attempts (problem solving):
How many times have you tried the FACS?
Have you run a "No Primary" control?
Do you obtain the same results every time?
What steps have you altered?
The concentration of the Ab
Asked on Apr 03 2012
Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.
The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.
I would like to reassure you that ab31891 is tested and covered by our 6 month guarantee for use inflow cytometryand human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.
Reviewing this case, I would like to offer some suggestions to help optimise the results. I would also appreciate if you can confirm some further details:
1. I can recommend to aliquot and store the antibody at -20oC as soon as possible, as directed on the datasheet. this will ensure the antibody remains stable and that we can continue to provide our guarantee.
2. I would appreciate if it is possible to provide some data, including positive and negative controls.
3. I can suggest a permeabilization step would be required in this caseas ErbB2 is localized in the cytoplasm and nucleus. Try permeabilizing in 0.2% Triton for 10 minutes.
4. I would appreciate if you are able to provide any more details of the sample preparation? 'Squashing' the cells in PBS may damage them so that staining is affected, and the cells may not be fully dissociated to single cell suspension. I can recommend to try dissociating the cells in trypsin. There is further information on sample preparation on the following webpage:
To ensure the sample is of good quality, a viability cell count in trypan blue can be tried. We recommend there should be over 95% viable cellsin the sample to ensure the sample is healthy enough to proceed with staining and givegood results.
5. This antibody will also detect ErbB2 in the mouse cells. and the CD326 antibody may detect CD326 in the mouse cells. Certainly for this ErbB2 antibody, it has not yet been tested in mouse, and may be cross reacting. It would be useful to includeahuman only positive control, such as SK-BR3 cells?
It would be beneficial to optimize the procedure on a known positive expressing cell line first.
6. What percentage of human Erb2 positive cells are you expecting in the tissue sample?
I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details including the results of the human positive control..
Answered on Apr 03 2012