Overview

  • Product name
  • Description
    Rabbit polyclonal to Rab5
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IF, IHC-P, ICCmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Cow, Human, Monkey
    Predicted to work with: Dog
  • Immunogen

    Synthetic peptide:

    PKNEPQNPGANSARGR

    conjugated to KLH, corresponding to amino acids 182-197 of Human Rab5 (Rab5A)

  • Positive control
    • Recombinant Human Rab5 protein (ab62956) can be used as a positive control in WB. Mouse or Rat Brain Tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab13253 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/2000. Detects a band of approximately 26 kDa (predicted molecular weight: 25.8 kDa).
ICC/IF Use at an assay dependent concentration. PubMed: 19348413
IHC-P 1/100.

Use Bouin's fixative to fix tissue.

ICC Use at an assay dependent concentration.

Target

Images

  • Confocal microscopy of fixed primary cultures of rat hippocampal neurons (embryonic day 18) showing immunocytochemical labelling of polyclonal rabbit anti-Rab5 (Abcam ab13253, 1/200; Alexa Fluor 488; 1/200; green) and monoclonal mouse anti-tubulin-II (Alexa Fluor 568 1/200; red). Magnification 63x; insert 180x.
  • ab13253 Rabbit polyclonal to Rab5 was used in in fixed HEK cells that had been transfected with Rab5-GFP Fluorescence microscopy of fixed Rab5-transfected HEK cells showing (left to right) immunocytochemical labelling of rabbit polyclonal Rab5 (ab13253, 1/500; Cy3; red), and Rab5-GFP (green) with their overlay (far right). Scale bar = 7µm. Showing good co-localisation.

  • ab13253 at 1/100 dilution staining Rab5 in mouse back skin tissue section by Immunohistochemistry (Bouin's fixative fixed paraffin-embedded tissue section). Antigen retrieval was done by microwave in citrate buffer. A Fluorophore-conjugated goat anti-rabbit secondary was used at 1/50 dilution.

  • Immunofluorescence analysis of HeLa cells, staining Rab5 (green) with ab13253.

    Cells were incubated for 30 min with 0.5 µM biotinylated transportan and fixed with PLP fixative. Cells were permeabilized and blocked with 0.01% saponin and 0.1% BSA for 8 minutes, before incubating with primary antibody. An AlexFluor®488-conjugated anti-rabbit IgG was used as the secondary antibody.

References

This product has been referenced in:
  • Kim T  et al. Timely regulated sorting from early to late endosomes is required to maintain cerebellar long-term depression. Nat Commun 8:401 (2017). Read more (PubMed: 28864821) »
  • Altadill T  et al. Enabling Metabolomics Based Biomarker Discovery Studies Using Molecular Phenotyping of Exosome-Like Vesicles. PLoS One 11:e0151339 (2016). WB . Read more (PubMed: 26974972) »
See all 22 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Thank you for contacting us.

The antibody ab13253 is guaranteed for ICC/IF in mouse cells; this means this product comes with fully money back guarantee if used as stated on the datasheet.

We unfortunately do not have any mouse specific image however we do have tested this with mouse cells so please buy the ab without any hesitation.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - other (HeLa)
Loading amount
100000 cells
Specification
HeLa
Gel Running Conditions
Reduced Denaturing (7.5%)
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Mar 31 2009

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.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Rat Cell (Hippocampal neuron culture)
Specification
Hippocampal neuron culture
Fixative
Paraformaldehyde
Blocking step
BSA as blocking agent for 40 minute(s) · Concentration: 5%

Dr. Randal Moldrich

Verified customer

Submitted Dec 02 2005

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Human Cell (human Rab5-transfected HEK cells)
Specification
human Rab5-transfected HEK cells
Fixative
Methanol
Blocking step
BSA as blocking agent for 40 minute(s) · Concentration: 5%

Dr. Randal Moldrich

Verified customer

Submitted Dec 02 2005

Question

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.

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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.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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