Donkey Anti-Rabbit IgG H&L (FITC) preadsorbed (ab97084)

We used the following protocol with Anti-Glucose Transporter GLUT4 antibody (ab62375):

NSO cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab62375, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (https://www.abcam.com/index.html?datasheet=96899) at 1/500 dilution for 30 min at 22°C.

Isotype control antibody was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

This antibody also gave a positive signal in NSO cells fixed with 4% paraformaldehyde (10 min) permeabilized with 0.1% PBS-Tween used under the same conditions.

For both of these methods permiabilisation was used.

I hope this information has been of help. If you require any further information, please do not hesitate to contact us again.

Thank you for contacting us in regards to pre-incubating a primary and secondary together prior to performing FACS analysis.

I have spoken to my colleague who is quite experienced in using flow cytometry and she has informed me that whilst pre-incubation can be performed, it can sometimes lead to the formation of particles which can be picked up as an event in themselves.

If you would like to avoid using a secondary antibody altogether we have a range of conjugation kits which would allow you to conjugate your primary antibody directly. More information on these kits can be found from the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=13148

If contemplating this please do consult the following guide to decide if the primary antibody you wish to use is compatible:

https://www.abcam.com/index.html?pageconfig=resource&rid=13156

If you would like any assistance in determining this, could you please let me know the primary antibody again (unfortunately I think I took down the wrong number when speaking with you) and I can have a look into this.

I hope this information has been of some help. If you require any further information please do not hesitate to contact us again.

Thank you for contacting us.
I am sorry to hear that you received so far no staining with the antibodies.

As we discussed by phone, I'd suggest trying the following:

1) try another antigen retrieval step (e.g. Tris/EDTA, pH 8)
2)use more priamry antibody
3) try using an HRP-conjugated secondary antibody and hematoxyline (for nuclear staining)- to check if any signal can be obtained at all, with the experimentalsetup you have.

Right now it is difficult to determine where the problem might stem from - there could be multiple causes. Thus, please let me know what results you obtained after trying these tips and we can discuss this further.

I wish you good luck and look forward to hear back from you.

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Thank you very much for contacting us.

Please find attached the QC ICC/IF image for this antibody. The cell line is A172 cells. The antibodies used is at 5ug//ml and detected using Alexa Fluor 488 conjugated secondary antibody. The nuclei are stained with DAPI.

Please note the protein location is membranous as well as cytoplasmic so in flow cytometry you will need a permabilzation step.

I hope this information will be helpful. Should you have any further question please do not hesitate to ask.