Overview

  • Product name
    Anti-KRAS+HRAS+NRAS antibody
    See all KRAS+HRAS+NRAS primary antibodies
  • Description
    Mouse monoclonal to KRAS+HRAS+NRAS
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant fragment (GST-tag) corresponding to Human KRAS+HRAS+NRAS aa 16-125.
    Sequence:

    KSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYS AMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVG NKCDLPSRTV


    Database link: P01116

Properties

Applications

Our Abpromise guarantee covers the use of ab55391 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 Use a concentration of 1 - 5 µg/ml. Predicted molecular weight: 22 kDa.
IHC-P Use at an assay dependent concentration.
Flow Cyt Use 0.5-1µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

 

Target

  • Relevance
    Ras proteins bind GDP/GTP and possess intrinsic GTPase activity. Plays an important role in the regulation of cell proliferation. Enzyme regulation: Alternates between an inactive form bound to GDP and an active form bound to GTP. Activated by a guanine nucleotide-exchange factor (GEF) and inactivated by a GTPase-activating protein (GAP). Interaction with SOS1 promotes exchange of bound GDP by GTP.
  • Cellular localization
    Cell Membrane, Cytoplasmic and Golgi Apparatus
  • Database links
  • Alternative names
    • c-H-ras antibody
    • c-K-ras antibody
    • c-Ki-ras antibody
    • GTPase HRas antibody
    • GTPase KRas antibody
    • GTPase NRas antibody
    • H-Ras-1 antibody
    • Ha-Ras antibody
    • HRAS1 antibody
    • K-Ras 2 antibody
    • Ki-Ras antibody
    • KRAS antibody
    • KRAS2 antibody
    • NRAS antibody
    • p21ras antibody
    • RASK2 antibody
    • Transforming protein N-Ras antibody
    • Transforming protein p21 antibody
    see all

Images

  • cK Ras antibody (ab55391) at 1ug/lane + HeLa cell lysate at 25ug/lane.
  • Overlay histogram showing HeLa cells stained with ab55391 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab55391, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in HeLa cells fixed with methanol (5 min)/permeabilized with 0.1% PBS-Tween 20 used under the same conditions.

  • ab55391 staining KRAS+HRAS+NRAS in Human Gastric Acenocarcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with 10% phosphate buffered formalin, permeabilized with 0.025% of triton X -100 in TBS and blocked with 3% hydrogen peroxide for 5 minutes at room temperature; antigen retrieval was by heat mediation in target retrieval solution. Samples were incubated with primary antibody (1/100 in 0.025% triton X-100-TBS) for 1 hour. An HRP polymer was used for detection.

References

This product has been referenced in:
  • Zhang L  et al. Loss of ARID1A Expression Correlates With Tumor Differentiation and Tumor Progression Stage in Pancreatic Ductal Adenocarcinoma. Technol Cancer Res Treat 17:1533034618754475 (2018). Read more (PubMed: 29486633) »
  • Bäumer N  et al. Downregulation of PIK3CA via antibody-esiRNA-complexes suppresses human xenograft tumor growth. PLoS One 13:e0200163 (2018). Read more (PubMed: 30001368) »
See all 16 Publications for this product

Customer reviews and Q&As

1-10 of 15 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Gel Running Conditions
Reduced Denaturing (12.5%)
Loading amount
1e+006 cells
Specification
HeLa
Blocking step
(agent) for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Jan 12 2016

Answer

The immunogen for this antibody is KRAS (NP_004976, 16 a.a. ~ 126 a.a). As we don't do epitope mapping, we can't provide you the exactly epitope where the antibody is targeting on. Therefore without testing, we cannot be sure if the mutated form of KRAS which you are studying will be recognized. Generally an epitope is 8-16 aa in length and often a single change in the aa sequence will not affect binding. Therefore it may be likely that this will recognize your antigen provided the antigen has sequence similar to the WT KRAS.

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Question
Answer

Thank you for contacting Abcam earlier today.

I am sorry that you were having some issues with ab55391. The antibody is covered under our Abpromise for six months and is guaranteed to work in IHC-P on human samples . Based on the protocol information that you provided, I do not think that protocol tips would help resolve this situation, therefore I would be happy to either send a replacement antibody or to process a refund.

Please let me know how you would you like to continue and if you could also provide either the Abcam order number or the PO# used to purchase the antibody, that would be very helpful.

I look forward to your reply.

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Answer

Merci pour ces précisions.

Comme convenu j'ai mis en place l'envoi d'une unité d'anti-KRAS ab55391. Le numéro de commande de remplacement gratuit de ce produit est *****. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

N'hésitez pas à nous contacter lors d'une prochaine occasion.

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Answer

Thank you for contacting us.

The current in stock lots are GR47401-1, GR47401-2.

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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Answer

Thank you for contacting Abcam.

Under our Abpromise we guarantee that all of those antibodies you listed would work on human tissue in IHC-P. However I would recommend ab55391, as It has protocol information available on the website.

If you wish to use the antibody in a different species of if I can help you select any other reagents you may need, please let me know.

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Question

1) Code du produit Abcam
ab84573
2) Numéro de commande Abcam ou le numéro de lot/batch
HS product code: *********** lot GR10593
3) Description du problème
Aucun marquage Kras n’est observable
4 Préparation de l’échantillon :
Espèce : Humain et Souris
Type d’échantillon : fixée paraffine,
Préparation de l’échantillon:
Contrôle positif: Oui
Contrôle négatif: Oui

5) Etape de fixation: Oui
Si oui : agent fixateur et concentration : PFA à 4%
Temps de fixation: 5H
Température de fixation: 4°C

6) Méthode pour l’antigen retrieval: bain au microonde 2x5minutes dans Tampon citrate 10mM (ph6)

7) Etape de perméabilisation :
Avez-vous procéder à une étape de permabilisation (à détailler) ou avez-vous additionné un agent de perméabilisation dans plusieurs tampons de dilution ?
Agent de perméabilisation et concentration
Perméabilisation faite avec bain 5 minutes sur agitation dans Tampon PBS-Triton X100 0,3%

8) Agent bloquant (ex BSA, serum…): BSA et lait
Concentration: BSA à 10% et lait à 3%
Temps de saturation : 45 minutes
Température de saturation: room température (25°C)

9) Blocage des peroxidases endogènes ? Non
Blocage des biotines endogènes ? Non

10) Anticorps primaire (utilisation de plusieurs anticorps, description dans “notes supplémentaires”) : Anti-Kras
Concentration et dilution: à 0,5ug/ml 1/200
Tampon de dilution PBS, 10 % BSA, 0,3% lait
Temps d’incubation Overnight

11) Anticorps secondaire : Anticorps Biotynilé
Espèce: sheep
Réagit contre: Rabbit
Concentration ou dilution 2ug/ml 1/500
Tampon de dilution PBS, 10% BSA
Temps d’incubation 1H
Température d’incubation 37°C
Fluorochrome or enzyme conjuguée

11bis)Anticorps tertiaire: Anti-biotin-POD, 1/200
Tampon de dilution: PBS, 10% BSA
Temps d’incubation: 1H

12) Lavage après les anticorps primaire et secondaire:
Tampon: PBS-Triton X100 0,1%
Nombre de lavage: 3

13) Méthode de détection DAB, pendant 1 minute

14) Combien de fois avez vous essayer cette application ? 2 fois

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Answer

Merci d'avoir pris le temps de remplir notre questionnaire et de nous avoir contactés. Je suis désolé d'apprendre que vous avez eu des difficultés à obtenir de bons résultats avec ab84573.

Notre politique est d'offrir tout d'abord la meilleure assistance technique. Si nous concluons que le produit que vous avez reçu ne fonctionne pas comme mentionné sur sa fiche technique, nous nous ferons un plaisir de vous offrir un remplacement gratuit avec une unité de ab55391.

J'aimerais donc vous offrir quelques suggestions afin d'optimiser vos résultats :

- Pour le démasquage de l'antigène, nous recommandons d'utiliser un tampon Tris/EDTA pH 9.0. Ce tampon, moins utilisé que le citrate pH 6.0,donnepour la majorité des anticorps de bien meilleurs résultats que le tampon citrate pH 6.0. Une publicationintéressante appuyant ce qui précède est : Emoto et al.,
J Histochem Cytochem. 2005 53 (11) : 1311-21.

J'espère que ces informations vous aideront. Si nos suggestions ne permettent pas d'améliorer vos résultats, je vous ferai parvenir une unité de ab55391 en remplacement.

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Answer

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibodyhas not been providing satisfactory results despite the protocol tips provided.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial. I apologize for the inconvenience and am pleased to offer you a free of charge replacement. Unfortunately we do not currently have any alternative lot in stock of ab55391 but I can offer you a different antibody such as https://www.abcam.com/KRAS-antibody-ab84573.htmlor https://www.abcam.com/KRAS-antibody-ab102007.html. These are both rabbit polyclonal antibodies to KRAS which has been used to perform western blotting with human samples.

Alternatively if preferable I can provide you with acredit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Answer

Thank you for clarifying the situation. Having reviewed the results and the protocol used I would recommend the following:


1.) Although I have found reference of KRAS expression in some of the used cell lines. I would still suggest using HeLa cell line if you have access to it as this is what has been used to characterize the antibody. With the other cell lines used I would increase the amount of protein loaded, up to ˜80 umicrog.


2.)I would like to recommend toalsouse analternative blocking buffer. Iwould recommend trying 5% BSA . As you may know, changing the blocking buffer can significantly change the results as illustrated on the WB image on the datasheet of ab9385 for example.

Additionally, could you confirm if the secondary anti mouse antibody is known to work (for example with other primary mouse antibodies)?

Many thanks for your cooperation.

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Question

            Dear technical support team: This customer has already purchased ab55391 (Anti-KRAS antibody), and after he conducted WB with human sample, the result shows weak signal, therefore he wants to ask for you help to offer any suggestion to him, I also attached the image in this letter, and his experiment step as follow:   1. Order details: Batch number: gr41950-1 Po: 976697 Abcam product code: ab55391 Antibody storage conditions (temperature/reconstitution etc)  -20C 2. Please describe the problem (high background, wrong band size, more bands, no band etc). No band and weak band 3. On what material are you testing the antibody in WB? ·         Species: human ·         What’s cell line or tissue SW480,SW620,HCT15,HCT116,HT29 ·         Cell extract or Nuclear extract: Intron biotechnology PRO-PREP protein extraction solution (EDTA free) ·         Purified protein or Recombinant protein: total protein   3.  The lysate How much protein was loaded: 35ug What lysis buffer was used: What protease inhibitors were used: What loading buffer was used: SDS-PAGE Phosphatase inhibitors Did you heat the samples: temperature and time:  95C/5 min   4.  Electrophoresis/Gel conditions/ Transfer conditions Reducing or non reducing gel: Reducing agent: Gel percentage : 10% and 12% Transfer conditions: (Type of membrane, Protein transfer verified): PVDF/semi dry   5. Blocking conditions Buffer: TBST Blocking agent: milk, BSA, serum, what percentage: milk 5% Incubation time: 1hr Incubation temperature:25C   6. Primary Antibody Species: human Reacts against: mouse ·         At what dilution(s) have you tested this antibody: 1:200 ·         What dilution buffer was used: TBST ·         Incubation time: over night (>16hr) ·         Incubation temperature:  4C ·         What washing steps were done: 1 time/5 min, 3 times   7. Secondary Antibody Species: Reacts against: mouse At what dilution(s) have you tested this antibody: 1:5000 Incubation time: 1hr Wash steps: 1 time/5 min, 3 times Fluorochrome or enzyme conjugate: Do you know whether the problems you are experiencing come from the secondary?  No.   8. Detection method ECl, ECl+, other detection method: ECL   9. Did you apply positive and negative controls along with the samples? Please specify.  No, but all cell-line should be positive 10. Optimization attempts ·         How many times have you tried the Western? 2 ·         Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):  YES, I have run a no primary control ·         Do you obtain the same results every time e.g. are background bands always in the same place?  Yes ·         What steps have you altered?  Concentration of primary antibody   Could you please help this customer to solve the problem? Thanks for your kindly help Best regards  

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Answer

Thank you for taking time to liaising with the customer and to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab55391.  I would also appreciate if you can confirm some further details: 1.) I can recommend to reduce the samples and to add proteinase inhibitors if this has not been done so yet. Indeed, the reduction of the samples with either DTT or bME can free the target protein from an eventual complex and make it available for detection. I do not know whether the KRAS protein is in a complex, the ab55391 has however been tested on reduced samples. Also adding the protease inhibitors will avoid eventual degradation. 2.) Can you confirm that the secondary anti mouse antibody is known to work (for example with other primary mouse antibodies)? 3.) I am not aware of the level of KRAS expression in the used cell lines. If the amount is not know, it might be useful to increase the amount of cell lysate loaded up to 60ug or to use HeLa cell lysate as positive control. 4.) Finally I would like to recommend to try also an alternative blocking buffer. I can recommend to try 5% BSA . As you know, changing the blocking buffer can significantly change the results as illustrated on the WB image on the datasheet of ab9385 for example.   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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1-10 of 15 Abreviews or Q&A

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