Overview

  • Product name
    Anti-MRP4 antibody [M4I-10]
    See all MRP4 primary antibodies
  • Description
    Rat monoclonal [M4I-10] to MRP4
  • Host species
    Rat
  • Tested applications
    Suitable for: IHC-P, ICC/IF, WB, IHC-Fr, ICCmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    This antibody was selected after immunization with a fusion protein containing the E. coli maltose binding protein and a fragment of the human MRP4 protein corresponding to amino acids 372-431.

  • Positive control
    • Kidney tissue This antibody gave a positive result in IHC in the following FFPE tissue: Human lung adenocarcinoma.

Properties

Applications

Our Abpromise guarantee covers the use of ab15602 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
IHC-P Use a concentration of 10 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use at an assay dependent concentration. PubMed: 22272278PFA fixed cells
WB 1/20 - 1/50. Predicted molecular weight: 159 kDa.
IHC-Fr 1/20. Fix with acetone.
ICC 1/20 - 1/50. (acetone fixed cytospin preparations)

Target

  • Function
    May be an organic anion pump relevant to cellular detoxification.
  • Tissue specificity
    Widely expressed, with particularly high levels in prostate, but is barely detectable in liver.
  • Sequence similarities
    Belongs to the ABC transporter superfamily. ABCC family. Conjugate transporter (TC 3.A.1.208) subfamily.
    Contains 2 ABC transmembrane type-1 domains.
    Contains 2 ABC transporter domains.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • ABCC 4 antibody
    • ABCC4 antibody
    • ATP binding cassette sub family C (CFTR/MRP) member 4 antibody
    • ATP binding cassette sub family C member 4 antibody
    • ATP-binding cassette sub-family C member 4 antibody
    • bA464I2.1 (ATP binding cassette, sub-family C (CFTR/MRP) member 4) antibody
    • bA464I2.1 antibody
    • Canalicular multispecific organic anion transporter antibody
    • Canalicular multispecific organic anion transporter ABC superfamily antibody
    • EST170205 antibody
    • MOAT B antibody
    • MOAT-B antibody
    • MOATB antibody
    • MRP 4 antibody
    • MRP/cMOAT related ABC transporter antibody
    • MRP/cMOAT-related ABC transporter antibody
    • MRP4_HUMAN antibody
    • Multi specific organic anion transporter B antibody
    • Multi-specific organic anion transporter B antibody
    • Multidrug resistance associated protein 4 antibody
    • Multidrug resistance-associated protein 4 antibody
    • OTTHUMP00000018560 antibody
    see all

Images

  • All lanes : Anti-MRP4 antibody [M4I-10] (ab15602) at 20 µg/ml

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : ABCC4 (MRP4) knockout HAP1 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 159 kDa



    Lanes 1 - 2: Merged signal (red and green). Green - ab15602 observed at 200-250 kDa. Red - loading control, ab176560, observed at 50 kDa.

    ab15602 was shown to specifically react with MRP4 in wild-type HAP1 cells as signal was lost in ABCC4 (MRP4) knockout cells. Wild-type and ABCC4 (MRP4) knockout samples were subjected to SDS-PAGE. Ab15602 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 20 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rat IgG H&L (IRDye® 800CW) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • HC image of MRP4 staining in Human lung adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab15602, 10µg/ml, for 15 mins at room temperature. A Goat anti-Rat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ab15602 staining MRP4 (green) in early cobblestone Human ESC-RPEs by Immunocytochemistry/ Immunofluorescence.

    Cells were fixed with paraformaldehyde and blocked with 3% BSA in PBS for 1 hour at room temperature; antigen retrieval was by 0.1% Triton X-100/PBS. Samples were incubated with primary antibody (1/100 in 0.5% BSA-PBS) for 2 hours at room temperature. An AlexaFluor®488-conjugated goat anti-rabbit IgG (1/1500) was used as the secondary antibody.

References

This product has been referenced in:
  • Sekhar GN  et al. Organic cation transporter 1 (OCT1) is involved in pentamidine transport at the human and mouse blood-brain barrier (BBB). PLoS One 12:e0173474 (2017). Read more (PubMed: 28362799) »
  • Chen JJ  et al. MRP4 regulates ENaC-dependent CREB/COX-2/PGE2signaling during embryo implantation. Oncotarget 8:78520-78529 (2017). Read more (PubMed: 29108246) »
See all 18 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

In order to find the secondary antibody that best fits your necessities; I would like you to provide us with further information, such as in which application are you testing the antibody or the host species for the secondary.
However, you can also do your own secondary antibody search through our website, and choose the best option for your experiments. To do it, go to our web site www.abcam.com.
Here you can search the antibody by choosing “Advance Search” from the Tab “Secondary Antibodies” from the scroll down menu in the Yellow bar at the top of the page. By filling the required fields, you will be lead to the secondary antibody results. (See hyperlink below).
https://www.abcam.com/index.html?pageconfig=productmap&cl=918
If you need any help with the search, do not hesitate to contact us.
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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Question

Reviewing this case, I would like to offer some suggestions to help optimise the results from these antibodies. I would also appreciate if you can confirm some further details:
1. Please confirm the order numbers and dates of purchase.
Order reference number: #####

Dates: 06/08/2012
2. Could you confirm which lysis buffer was used? I can recommend to try RIPA buffer if not already tried. This should provide a suitable protein preparation.
The lysis buffer was a fixed kit, we bought from ThermoFischer to isolate membrane proteins and a buffer based on EDTA.
3. We recommend samples should be reduced and denatured and run on a reducing gel in WB unless stated otherwise on the datasheet. This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody.
The samples were reduced and diluted with the Laemmli buffer. Up to now I never used a reducing gel for my other ABC transporters and it always worked.
4. I can recommend to consider trying BSA rather than milk to block. Changing blocking agent can sometimes help to improve results.
This I will try
5. Could you confirm if the transfer to the membrane and quality of the samples been assessed using a loading control?
A coloured marker was used, showing a positive transfer onto the blot. A loading control was not used.
6. Are the current vials of secondary antibody and detection kit working well with other primary antibodies?
The detection kit works with other primary antibodies, the second antibody is new and didn't worked up to now. Can you maybe suggests an other working second antibody for these antibodies, I need with FITC labelling for IHC too.

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Answer

Thank you for your reply and for kindly providing the requested details.

I look forward to hearing from you with the results of the next experiment. i can recommend it would be beneficial to consider a reducing gel. This will ensure the protein is in the correct conformation for detection by the antibody. Although the other antibodies work well using a non reducing gel, individual antibodies detect individual epitopes and so will sometimes require individual optimization.

I hope this will be helpful. Please do not hesitate to let me know how you get on with the reducing gel. I look forward to hearing from you.

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Question

Thank you for your time and cooperation. I look forward to receiving the completed questionnaire.
Order Details
Antibody code:
MRP4: ab15602, GR79982-1
MRP5: ab 3377, GR43649-3
MRP2: ab 3373, GR19771-1
Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background
No signal in IHC for MRP4 and MRP5
Lot number
MRP4: ab15602, GR79982-1
MRP5: ab 3377, GR43649-3
MRP2: ab 3373, GR19771-1
Purchase order number
or preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
-20oC except MRP5, fridge
Description of the problem (high background, wrong band size, more bands, no band etc.)
No band

Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Cell extract

Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
Lyse Buffer with EDTA + Protease inhibitor cocktail

Amount of protein loaded
around 20 ug
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Non-reducing gel, 7.5% gel, running for 90 minutes, 150V, 80mA and 25W
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Buffer = without Methanol, with glycin and SDS for 2.5h with 320mA for two gels, blocking agent is a 2% Milkpowder in PBS+Tween20
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
MRP4: 1/50 dilution in milkpowder solution
MRP5: 1/50 dilution in milkpowder solution
MRP2: 1/100 dilution in milkpowder solution
Incubation in fridge overnight, washstep 3 times for 10 minutes with PBS+Tween20
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Secondary antibody is from calbiochem, againt the whole IgG fraction. Produced in mouse or rat
MRP4: 1/100 dilution in milkpowder solution
MRP5: 1/50 dilution in milkpowder solution
MRP2: 1/100 dilution in milkpowder solution
Incubation 1Hour at roomtemperature, washstep 3 times for 10 minutes with PBS+Tween20
Detection method (ECL, ECLPlus etc.)
Peroxidase, chemilumiscence
Positive and negative controls used (please specify)
My positive control is the caco2 cell line, in which no detection occurs, no negative controls were used
Optimization attempts (problem solving)
How many times have you tried the Western?
Two times
Have you run a "No Primary" control?
No
Do you obtain the same results every time?
Yes
e.g. are the background bands always in the same place?
There are no background bands
What steps have you altered?
Non
Additional Notes:
Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to assess the results.

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Answer

Thank you for taking the time to complete our questionnaire. The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimise the results from these antibodies. I would also appreciate if you can confirm some further details:

1. Please confirm the order numbers and dates of purchase.

2. Could you confirm which lysis buffer was used? I can recommend to try RIPA buffer if not already tried. This should provide a suitable protein preparation.

3. We recommend samples should be reduced and denatured and run on a reducing gel in WB unless stated otherwise on the datasheet. This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody.

4. I can recommend to consider trying BSA rather than milk to block. Changing blocking agent can sometimes help to improve results.

5. Could you confirm if the transfer to the membrane and quality of the samples been assessed using a loading control?

6. Are the current vials of secondary antibody and detection kit working well with other primary antibodies?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

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Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from these antibodies.

I am sorry to confirm that because we carry over 90,000 products, it is regrettably not feasible for us to stock small sample sizes of our products to send to customers. If you are concerned about the secondary antibody, I can recommend to consider contacting the supplier of the secondary. I notice that MRP4 antibody [M4I-10] (ab15602) is a rat IgG2a, do you know that the secondary antibody will detect this isotype?

ab3373 Anti-MRP2 antibody [M2 III-6] should be working in WB, it has been tested successfully. Particularly if it has worked in the IHC experiments which shows the antibody is active, it is unusual this is not working in WB. Again, this is a mouse IgG2a isotype. Will the secondary antibody used in WB detect this, was this the same secondary used in IHC-P?

I would like to reassure you that all these antibodies are tested and covered by our 6 month guarantee for WB and the species that are listed on the individual datasheets. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

Thank you for your time and cooperation. I look forward to receiving the completed questionnaire.


Order Details
Antibody code:
Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background
Lot number
Purchase order number
or preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
Description of the problem (high background, wrong band size, more bands, no band etc.)
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
Amount of protein loaded
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Detection method (ECL, ECLPlus etc.)
Positive and negative controls used (please specify)
Optimization attempts (problem solving)
How many times have you tried the Western?
Have you run a "No Primary" control?
Yes No
Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?
What steps have you altered?
Additional Notes:
Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to assess the results.

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Question
Answer

Thank you for your enquiry. The publication found at the following link contains images of Western blots for both of these antibodies: http://mcb.asm.org/cgi/content/full/24/17/7612?view=long&pmid=15314169 I hope this helps. Please contact us if you have any more questions.

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Answer

Thank you for your enquiry. I am sorry for the delay to your enquiry; I have been in touch with the source of this antibody and a response has not been forthcoming. For the testing of antibodies ab15598 and ab15602 against MRP4 I have been informed that HEK-cells transfected with MRP4 can be used. Furthermore MRP4 can be detected in HEK-cells alone. They have also provided me with the recommendation of kidney tissue. With regards ab3377 and a positive control lysate I am sorry but I have not been able to source information such that I am enable to suggest a positive control lysate. I apologise for this. I would like to suggest that you perform a search of the literature. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us for technical support with ab15602. I am currently trying to find out the recommended positive control for this antibody and as soon as I have this information I will let you know. The antibody is supplied as tissue culture supernatant therefore the recommended dilutions are in the range of 1:20-50 (overnight incubation), please make sure you dilute in TBS Tween20 0.1% and incubate at 4C. The problem may also be due to poor extraction of the protein from the cell membrane, please make sure you use a strong detergent (e.g NP40) in the lysis buffer containing protease inhibitors. Without further protocol details it is very hard to know what the problem might be and if the customer still experiences problem please guide him to the link below where a questionnaire will help him to put all the details easily together and we can investigate this matter in details, https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=15602&mode=questionaire Please do not hesitate to contact me if you require further help,

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Answer

Thank you for your enquiry regarding ab15602. This antibody has not been tested in rat (only human and mouse) but we would not advise to use it in rat in IHC as the antibody has been raised in rat and hence the secondary antibody anti-rat is very likely to bind to all the rat tissue and give non specific binding. I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

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