Anti-Rab5 antibody - Early Endosome Marker (ab13253)

I am seeing nuclear staining with this antibody, when it is listed as being localized at the cytoplasmic surface of the plasma membrane. Have you seen nuclear staining with this antibody before?

ANSWER:

Thank you for your patience, and I apologize for the delay in answering your question.

We have not personally tested this antibody for IHC/IF but it has been cited in a publication: http://www.jbc.org/cgi/content/full/276/21/18540

As one would expect with Rab5, the staining in the publication is localized to the membrane region of the tissue. The protocol used is well detailed in the publication, including their antigen unmasking technique. It is possible that you will get better staining by optimizing your procedure a bit. The nuclear staining may just be non-specific.

I hope this information helps. Please do not hesitate to contact us if you need anything further.

BATCH NUMBER 74988

DESCRIPTION OF THE PROBLEM High background. No signal

SAMPLE Cos 7 cells, whole cell, light mitochondrial pellet or subcellular fractions.

PRIMARY ANTIBODY rabbit anti Rab5 abcam, 1:2000

SECONDARY ANTIBODY goat anti rabbit, peroxidase coupled, Jackson

DETECTION METHOD ECL, amersham

POSITIVE AND NEGATIVE CONTROLS USED positive control: I saw a band for Rab 5 once with whole cell lysate. All other attempts with this antibody resulted in no signal after shorter exposures or black blots after longer exposures. negative control: only secondary antibody gives no background other positive controls: a rabbit anti calreticulin antibody I use works fine. The same secondary antibody is used by many other people in the lab and works fine. All the other antibodies I use (mouse anti Tom22, anti cyt c oxidase, NaK ATPase, EEA1, LAMP-1; rat anti HA) work great without background.

ANTIBODY STORAGE CONDITIONS Arrived at RT. Aliquoted on day of arrival. 10 micro liter aliquots stored at -20. Used aliquots stored at 4 degrees.

SAMPLE PREPARATION Differential Centrifugation - 3 60mm Petri dishes of COS 7 cells - rinse cells once with cold PBS, scrape cells into 2ml cold PBS per dish - spin cells down 1min on setting 4 - resuspend pellet in 500microl cold HM, transfer to Eppendorf vial, spin 1min at 5000g - take off supernatant, resuspend pellet in 100microl cold HM + complete - transfer to cold Dounce homogenizer, homogenize with 25 strokes using pestle B - add 300microl cold HM + complete, transfer to Eppendorf tube, spin 10min at 1000g, 4?C - save supernatant, resuspend pellet in 100microl cold HM + complete - transfer to cold Dounce homogenizer, homogenize with 25 strokes using pestle B - add 300microl cold HM + complete, transfer to Eppendorf tube, spin 10min at 1000g, 4?C - take off supernatant, combine supernatants, spin 10min at 1000g, 4?C - take off supernatant, spin 10min at 1000g, 4?C - take off supernatant, spin 10min at 17,000g, 4?C -> resuspend pellet in 1.5ml 30% OptiPrep, save 250microl for Western - layer under OptiPrep gradient - centrifuge 3 hours at 52,000gav (20 500rpm for TH-641 rotor) in OptiPrep gradient. - collect 13 drop fractions - mix each fractions with 4x SDS sample buffer (with BME) - load 15 microl of each fraction and of input for Western blot

AMOUNT OF PROTEIN LOADED Not determined.

ELECTROPHORESIS/GEL CONDITIONS 9% SDS Tris gels. BME as reducing agent.

TRANSFER AND BLOCKING CONDITIONS NaCO3/NaHCO3 buffer with 10% methanol. PVDF membrane. Block: 1% milkpowder in TBS + 0.5% Tween. 1h at RT or over night at 4 degrees.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? - higher and lower secondary concentration (1:2000, 1:5000, 1:10,000) - more washes - longer and shorter exposure times (20s to 1 hour)

ADDITIONAL NOTES I did have similar problems with the rabbit anti-calreticulin antibody I use. But this problem was solved by altering the secondary concentration.

ANSWER:

Thank you for your enquiry and for taking your time to fill in the on-line questionnaire. We are very sorry to hear that you are having problem with this antibody.

According to our order record and the database, we have sold several vials of this batch number without any problem. Therefore, at this stage, we would suggest that there is either a problem with the vial you received, or modifications to your protocol are needed to obtain a positive result.

We would strongly recommend determining the protein concentration of the samples and loading at least 25 or 30 ug per lane. Otherwise you can’t be sure that you have loaded enough material onto the gel.

The recommended dilution for Western blot is 1/2000 if sufficient amount of protein is loaded. Since you have not determined the concentration, you may need to increase the concentration of the primary antibody (1/500 and 1/1000) to see if the signal is getting stronger.

You have not mentioned how long you incubated the membrane with the primary antibody. You can also try to apply overnight incubation at 4 oC or 2 hrs at room temperature.

It may also be worth running positive control along with the samples. This antibody has been tested for Western blot application using mouse or rat whole brain tissue extract.

Try to block the nonspecific bindings with 5% BSA or 5% nonfat dry milk instead of 1% milk.

We hope this will help. Should you still have any problem with this product, then please do not hesitate to contact us again.