Question (63383) | Anti-E. coli LPS antibody [2D7/1] (ab35654)

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Question



Inquiry: Hello, We used your Anti-E. coli LPS antibody [2D7/1] to detect LPS on E. Coli however using epifluorescence microscopy, we were not able to detect any signal. We first fixed two different strains of E. coli (J5 and o111, a pellet from 50ul of overnight growth ) with 4% paraformaldehyde (in 1X PBS). The fixed pellets were then each resuspended in 100ul 1X PBS. We then added a concentration of 1:100 of the anti-LPS to each strain, and incubated the samples for 30 min at room temperature. Each strain was then washed 3 times with 500ul of 1X PBS and then a 1:100 concentration of the secondary antibody AF 488 rabbit, anti-mouse was added to each sample and incubated for 30 minutes. Each sample then was washed 3 times with 1X PBS and then placed on a slide and viewed under the microscope. We also made controls of secondary AF 488 only, a isotype IgG control, and an unstained sample, and were not able to detect any signal for any of the samples. We were wondering if you could tell us why we were not able to detect LPS signal, suggest any alternate way we could use to verify that the anitbody works, as well as if fixing the E. coli strains with paraformaldehyde affected the LPS-antibody interaction? Thank you

Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I am curious about the possibility that the secondary antibody is causing the problem. You state you used the IgG control and yet you did not any staining, which indicates it is not the primary antibody. Do you have another secondary antibody which you could try?
Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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