Anti-Rab5 antibody [Rab5-65] (ab50523)

ReviewID: XXXX AbID: 50523, Anti-Rab5 antibody [Rab5-65] Rating: Below Average Image: Anti-Rab5 antibody [Rab5-65] for Immunocytochemistry/ Immunofluorescence (Human) Sample: Species: Human Type: Cell Specification: HEK-T Application: Application: Fixative: Paraformaldehyde Permeabilization: No Blocking step: None as blocking agent for Other detail: Dilution: 1/100 Incubation time: 1 hour at 37°C Diluent: PBS-0.05%saponin Secondary Antibody: Name: Non-Abcam Antibody was used: donkey-anti-mouse Alexa488 Conjugation: Alexa Fluor® 488 Dilution: 1/300 Additional Data: Additional Notes: Vesicular, but a lot of nuclear background observed

ANSWER:

Thank you for sharing your results through our Abreview system. I am sorry this antibody did not provide the expected results.

Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered.

-Have you run a positive control in parallel with your samples to help find out the source of the failure? For this particular antibody, HeLa cells are recommended.

- Have you incubated the samples with this primary antibody for longer period of time such as overnight at 4oC?

- Have you tested different fixatives to see which the most optimal solvent to fix the cells is? You can fix and permeabilise as described in the ICC-IF image on the datasheet, try ethanol/methanol (10 min/1 min) at –20 °C.

- I can recommend including 0.2% Tween 20 in the wash buffer and antibody dilution buffer. This will help to keep the cells permeabilized and help to wash away any excess antibody.

-I would also suggest using a blocking agent like BSA to reduce the background signal.

- Does the secondary work with other primary antibodies? What result did you obtain from the no-primary control?

I copy the link to a general ICC protocol from our website that could be very helpful to optimise the results from this antibody:

http://www.abcam.com/index.html?pageconfig=resource&rid=11417

I hope this information is helpful. Should the suggestions not improve the results, please do not hesitate to contact me again and I will try to provide further help.