Anti-Rab5 antibody - Early Endosome Marker (ab18211)

Thanks for your response. I was just wondering what temperature and for how long you would recommend fixing cells for with the acetone? Thanks

ANSWER:

I typically use acetone like methanol, by cooling it down in the freezer in a little pot (for 30min-1hr), then aspirating the medium of cells and adding the acetone on the cells and leaving them to be fixed on the bench (I would recommend trying 5 and 10 mins).

I hope this helps, please let me know how the next set of experiments go!

I have attached a file relating to my enquiry. Apologies for not attaching it previously - I did prepare the file to do so but then completely forgot to attach it! As you can se, with the Rab5 antibody I observe perinuclear/cytoplasmic staining however with the EEA1 antibody I observe punctate staining scattered across the cell surface - obviously these two patterns of localization differ despite the two antibodies being early endosome markers. Hope this helps Thanks for examining my images.

ANSWER:

Many thanks for the images, the Rab5 is particularly beautiful, it must be really nice looking down the microscope and seeing this staining! I think the Rab5 is working well, and we need to concentrate on why the EAA1 antibody is giving a different staining. I looked at the Abreviews for this antibody and there is an image from an anonymous researcher which looks very much like your image of EAA1 (and the reviewer has used formaldehyde fixation on mouse Hepa cells).

I am wondering if too much fixation or the wrong fixative alters the epitope and makes the antibody give non specific staining. You mention having done a no primary control, can I please check that this was for both antibodies? I would recommend to try the following: -fix the cells in methanol for 5 minutes with ice cold methanol (but fix the cells at room temperature) (it might be worth trying acetone too) -add less primary antibody (trying 1:500 for example), but maybe incubating longer so the antibody binds slowly but specifically -add less secondary antibody (I use Alexa antibodies at 1:2000-1:3000)

As Rab5 and EAA1 are not the same protein I am not surprised that they do not give an identical staining pattern in the cytosol, and I hope that the modifications to the protocol for EAA1 will remove the nuclear background staining you currently have.

Please do not hesitate to contact me if you still experience problems, with the above changes, I would be happy to offer you a refund on your order if you purchased the antibodies in the last 90 days,

DESCRIPTION OF THE PROBLEM Using the Rab5 antibody for IF in both mouse liver (Hepa) cells and mouse embryo fibroblasts (PEA13), I have observed a staining pattern consistent with the published picture on your website. However I also use your EEA1 antibody (ab2900) and whilst I obtain good staining with this - I notice that the staining pattern produced by the two antibodies is not the same. They are both supposed to be markers of early endosomes therefore I was wondering if you could explain their different staining patterns in untreated cells?

SAMPLE Mouse liver (Hepa) cells mouse embryo fibroblasts (PEA13s)

PRIMARY ANTIBODY anti Rab5 (ab18211)1 ug/ml in blocking buffers 1 h at RT anti EEA1 (ab2900) 1:200 in blocking buffers 1 h at RT Washed 3 x 5 mins TBS-T

DETECTION METHOD IF microscopy

SAMPLE PREPARATION Samples fixed using either methanol at -20 degC for 10 mins or 3.7% formaldehyde for 10mins followed by 0.1% Triton-X-100 for 5 mins.

TRANSFER AND BLOCKING CONDITIONS Cells blocked with either 10% NGS-PBS or 5% BSA-TBS-T

SECONDARY ANTIBODY 1:1000 AlexaFluor 488 goat anti-rabbit IgG diluted in blocking buffers 1 h at RT Washed 3 x 5 min TBS-T

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes

WHAT STEPS HAVE YOU ALTERED? Different blockers as mentioned above and different methods of fixation.

ANSWER:

Thank you for contacting us for technical support with ab2900 and ab18211.

It would be very useful if you could send images of your staining so we can better understand your data.

Thank you very much for sending those, I look forward to hearing from you,