Overview

  • Product name
  • Description
    Rabbit polyclonal to FLIP
  • Host species
    Rabbit
  • Specificity
    Recognizes all FLIP splice variants including FLIP alpha, beta, gamma, and delta.
  • Tested applications
    Suitable for: WB, ICC/IF, Flow Cyt, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human FLIP aa 2-19.
    Sequence:

    SAEVIHQVEEALDTDEKE


    (Peptide available as ab8457)

Properties

Applications

Our Abpromise guarantee covers the use of ab8421 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 1/500 - 1/1000. Predicted molecular weight: 55 kDa.Can be blocked with Human FLIP peptide (ab8457).
ICC/IF Use a concentration of 5 µg/ml.
Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

 

IHC-P Use a concentration of 5 µg/ml.

Target

  • Function
    Apoptosis regulator protein which may function as a crucial link between cell survival and cell death pathways in mammalian cells. Acts as an inhibitor of TNFRSF6 mediated apoptosis. A proteolytic fragment (p43) is likely retained in the death-inducing signaling complex (DISC) thereby blocking further recruitment and processing of caspase-8 at the complex. Full length and shorter isoforms have been shown either to induce apoptosis or to reduce TNFRSF-triggered apoptosis. Lacks enzymatic (caspase) activity.
  • Tissue specificity
    Widely expressed. Higher expression in skeletal muscle, pancreas, heart, kidney, placenta, and peripheral blood leukocytes. Also detected in diverse cell lines. Isoform 8 is predominantly expressed in testis and skeletal muscle.
  • Sequence similarities
    Belongs to the peptidase C14A family.
    Contains 2 DED (death effector) domains.
  • Domain
    The caspase domain lacks the active sites residues involved in catalysis.
  • Post-translational
    modifications
    Proteolytically processed; probably by caspase-8. Processing likely occurs at the DISC and generates subunit p43 and p12.
  • Information by UniProt
  • Database links
  • Alternative names
    • c FLIP antibody
    • c FLIPL antibody
    • c FLIPR antibody
    • c FLIPS antibody
    • c-FLIP antibody
    • c-FLIPL antibody
    • c-FLIPR antibody
    • c-FLIPS antibody
    • CASH antibody
    • CASP8 and FADD like apoptosis regulator antibody
    • CASP8 and FADD-like apoptosis regulator subunit p12 antibody
    • CASP8AP1 antibody
    • Caspase homolog antibody
    • Caspase like apoptosis regulatory protein antibody
    • Caspase related inducer of apoptosis antibody
    • Caspase-eight-related protein antibody
    • Caspase-like apoptosis regulatory protein antibody
    • Casper antibody
    • Cellular FLICE-like inhibitory protein antibody
    • CFLA antibody
    • Cflar antibody
    • CFLAR_HUMAN antibody
    • CLARP antibody
    • FADD like anti apoptotic molecule antibody
    • FADD-like antiapoptotic molecule 1 antibody
    • FLAME antibody
    • FLAME-1 antibody
    • FLAME1 antibody
    • FLIP antibody
    • I FLICE antibody
    • I-FLICE antibody
    • Inhibitor of FLICE antibody
    • MACH-related inducer of toxicity antibody
    • MRIT antibody
    • OTTHUMP00000163715 antibody
    • OTTHUMP00000206475 antibody
    • OTTHUMP00000206476 antibody
    • OTTHUMP00000206478 antibody
    • OTTHUMP00000206479 antibody
    • OTTHUMP00000206480 antibody
    • OTTHUMP00000206482 antibody
    • OTTHUMP00000207360 antibody
    • Usurpin antibody
    • Usurpin beta antibody
    see all

Images

  • Lane 1 : Anti-FLIP antibody (ab8421) at 1 µg/ml
    Lane 2 : Anti-FLIP antibody (ab8421) at 2 µg/ml

    All lanes : K562 cell lysate

    Predicted band size: 55 kDa

  • Immunohistochemistry of FLIP in mouse liver tissue with FLIP antibody at 5 µg/ml.

  • ab8421 at 5µg/ml staining FLIP in Hela cells by ICC/IF 

References

This product has been referenced in:
  • Padmanabhan C  et al. cFLIP critically modulates apoptotic resistance in epithelial-to-mesenchymal transition. Oncotarget 8:101072-101086 (2017). Read more (PubMed: 29254146) »
  • Hernandez L  et al. A dual role for Caspase8 and NF-?B interactions in regulating apoptosis and necroptosis of ovarian cancer, with correlation to patient survival. Cell Death Discov 1:15053 (2015). WB . Read more (PubMed: 28179987) »
See all 10 Publications for this product

Customer reviews and Q&As

1-9 of 9 Q&A

Answer

Thank you for getting back to me.

The peptide would be free, and it would be sent through Tecnolab. As it will be delivered by them I am unsure of how long it will take. The peptide is in stock and would be sent out as soon as possible. I will contact Tecnolab to enquire how long this will take to get to you.

If you have any further questions, please do not hesitate to contact us again.

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Answer

That is no problem. I'm glad you have found it useful.

Once you have received the peptide, please do let me know how you get on. Until then, I wish you all the best with your research.

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Answer

Sorry for the delay in getting back to you.

I have some further information to share with you in regards to anti-FLIP antibody (ab8421). I have attached the quality control western blots obtained using this antibody. As can be seen from the western blot, it seems that both the mouse and rat FLIP protein is detected somewhat lower than 55 kDa whilst the human protein is just above the 55 kDa marker. This is in agreement with the results you have obtained so far, particularly with your samples from the anterior pituitary. I think using the blocking peptide will provide a good control to see that a specific band is being detected.

Unfortunately, I have had difficulty in contacting Tecnolab in order to find out exactly when you might be expected to receive the blocking peptide. I will keep trying to contact them but it may be quicker for you to contact them directly to find out.

I hope this information has been of help. I will let you know as soon as I hear anything further.

Many thanks for your continued cooperation.

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Answer

Thank you for contacting me.

I have added the blocking peptide (ab8457), to the next Tecnolab order. Theyusually organise for monthly orders with us andI am still awaiting a response as to when this is likely to be delivered to you. I will get back to you as soon as I have further information to share with you.

I am very sorry for the delay and any inconvenience caused.

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Answer

Thank you for getting back to me with that information.

I do not have any reference with this protein in particular showing a certain shift, it is just with slightly different running conditions of gels there can be a difference in the size observed. An example of this is where ladders are run under different conditions, in the following link using Tris-Glycine, Bis-Tris (MOPS buffer) andBis-Tris 4-12% (MES Buffer) produces shifts in where the bands migrate.

https://www.abcam.com/Prism-Protein-Ladder-10-175-kDa-ab115832.html

Similar effects can be produced in slight differences in the sample preparation and what gels are used. I am not sure that this is the case with your results but as the band detectedseems quite clean (especially in the anterior pituitary samples) and close to where we would expect it to be, it is quite likely.

If you would like to determine thismore conclusively, I can offer to send you a blocking peptide of the antibody if you would like?

I look forward to recieving your reply.

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Question

Thanks for your answer. Below, WB questionaire is complete. Also, I attach imageof my blot.I wait for your suggestions.

Best regards,

WB Questionnaire
It will take approximately 5 minutes to complete the questionnaire.

Please fill-in all fields so that we can better assist you
1) Abcam product code AB8421
2) Abcam order reference number or product batch number LOT GR67534-1
3) Description of the problem:I don´t have the specific band of FLIPL (55kDa).
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysate
Lysis buffer: yes
Protease inhibitors: yes
Phosphatase inhibitors: yes
Reducing agent: yes.(2-mercaptoethanol)
Boiling for ≥5 min? yes
Protein loaded ug/lane or cells/lane: 30ug/lane
Positive control: yes. Proteins from ovary
Negative control: no
5) Percentage of gel: 12%
Type of membrane: PVDF
Protein transfer verified: yes
Blocking agent and concentration: 5% of milk
Blocking time: 90 min
Blocking temperature: room temperature
6) Was a fresh membrane used or had it been probed and stripped with a different antibody? Fresh membrane
7) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution: 1ug/ml, 2ug/mlor 4ug/ml
Diluent buffer: TBS 5% milk
Incubation time: over night
Incubation temperature: 4 ºC
8) Secondary antibody:
Species: goat
Reacts against: rabbit
Concentration or dilution: 0.3ug/ml
Diluent buffer: TBS 5% milk
Incubation time: 1 h
Incubation temperature: room temperature
Fluorochrome or enzyme conjugate: Peroxidase conjugated
9) Washing after primary and secondary antibodies:
Buffer: TBS
Number of washes: 3 (with agitation)
10)Detection method: chemiluminescence
11) How many times have you run this staining? 1. Only in one experiment I have run twice.
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? I used different samples and concentrations ofanti-FLIP antibody.

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Answer

Thank you for providing that information. It has greatly helped in my understanding of the problems encountered. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.


Could I just ask, what is the difference in the samples loaded in the left hand and right hand blots?

It appears that in both blots (particularly in the left hand blot), that the expected band may be detected (around 50 kDa). Differences in exactly where a band is observed can alter depending on how the protein gel is run and how the samples are prepared, so this kind of shift of less than 5 kDa is not unusual. I would however like to try and understand why the band at ˜150 kDa is detected in the second blot.

There are a few suggestions that I can make with may help to improve the results observed so far:

1. I would try using 5% BSA blocking instead of milk. This can sometimes lead to dramatic changes in the efficiency of some antibodies (demonstrated on the datasheet of https://www.abcam.com/ab9385). I would also use BSA in the antibody diluent used.

2. I would introduce 0.1% Tween into the buffers used (including blocking buffer, antibody diluent and wash buffers). Thiscan help to keep the antibody solvated and wash the membrane moreeffectively.

3. I would suggest continuing to usethe1/500 dilution as this seems to be producing the best results so far.

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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Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

Thank you for providing the results which you have obtained with anti-FLIP antibody ab8421. I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary.

If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

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Answer

Thank you for your phone call. I am very sorry to hear that these 3 antibodies are not providing satisfactory results. The details provided will enable us to investigate these 3 cases 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. As we discussed, I believe these 3 products should have given satisfactory results. It appears that you may have received either faulty vials, or the shipment was affected by temperature variations, customs or other unknown factors. I apologize very much for the inconvenience, but am pleased to let you know that I have arranged for the replacement shipment as we discussed. Thank you for your understanding. I wish you good luck with your research! Please let me know if there is anything else I can help you with.

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Answer

We do not have a blot image available. However, as you can see from the datasheet, it can be used in WB at a 1:500 to 1:1000 dilution and will recognise all FLIP splice variants including FLIP alpha, beta, gamma, and delta and is human, mouse and rat reactive. If the antibody does not work as specified on the dataheet we will offer a replacement vial or a full refund.

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