Anti-eIF4G1 antibody (ab2609)

Reviews (3)Q&A (10)References (16)

Overview

  • Product name
    Anti-eIF4G1 antibody
    See all eIF4G1 primary antibodies
  • Description
    Rabbit polyclonal to eIF4G1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IP, WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Rat, Human, African green monkey
    Predicted to work with: Mouse, Rabbit, Horse, Hamster, Cow, Cat, Dog, Chimpanzee, Rhesus monkey, Gorilla, Chinese hamster, Orangutan, Elephant
  • Immunogen

    Synthetic peptide (Human) - which represents a portion of human eukaryotic translation InitiationFactor 4 Gamma 1 encoded in part by exons 10 and 11.

  • Positive control
    • Rat liver lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab2609 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
IP 1/1000.
WB 1/1000 - 1/10000. Detects a band of approximately 200 kDa (predicted molecular weight: 220 kDa). EIF4G is more susceptible to degradation compared to other proteins, especially from some tissue sources such as liver. This is true even when tissue is stored at frozen. SDS-PAGE sample buffer may improve the stability, but samples that are stored frozen may show degradation bands that have been described in the literature.Therefore if multiple bands are observed in WB, it is likely due to the degradation of eIF4G rather than non-specificity of the antibody.
IHC-P Use a concentration of 4 µg/ml.
ICC/IF 1/500.

Target

  • Function
    Component of the protein complex eIF4F, which is involved in the recognition of the mRNA cap, ATP-dependent unwinding of 5'-terminal secondary structure and recruitment of mRNA to the ribosome.
  • Involvement in disease
    Defects in EIF4G1 are the cause of Parkinson disease type 18 (PARK18) [MIM:614251]. An autosomal dominant, late-onset form of Parkinson disease. Parkinson disease is a complex neurodegenerative disorder characterized by bradykinesia, resting tremor, muscular rigidity and postural instability, as well as by a clinically significant response to treatment with levodopa. The pathology involves the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (intraneuronal accumulations of aggregated proteins), in surviving neurons in various areas of the brain.
  • Sequence similarities
    Belongs to the eIF4G family.
    Contains 1 MI domain.
    Contains 1 MIF4G domain.
    Contains 1 W2 domain.
  • Post-translational
    modifications
    Phosphorylated at multiple sites in vivo. Phosphorylation at Ser-1185 by PRKCA induces binding to MKNK1.
    Following infection by certain enteroviruses, rhinoviruses and aphthoviruses, EIF4G1 is cleaved by the viral protease 2A, or the leader protease in the case of aphthoviruses. This shuts down the capped cellular mRNA transcription.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • DKFZp686A1451 antibody
    • eIF 4 gamma 1 antibody
    • eIF 4G 1 antibody
    • eIF 4G1 antibody
    • eIF-4-gamma 1 antibody
    • eIF-4G 1 antibody
    • eIF-4G1 antibody
    • EIF4 gamma antibody
    • EIF4F antibody
    • EIF4G antibody
    • EIF4G1 antibody
    • EIF4GI antibody
    • Eukaryotic translation initiation factor 4 gamma 1 antibody
    • IF4G1_HUMAN antibody
    • p220 antibody
    see all

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4G1 antibody (ab2609)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4G1 antibody (ab2609)
    ab2609 (4µg/ml) staining eIF4G1 in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of the cytoplasm of the intestinal cells.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • Immunoprecipitation - Anti-eIF4G1 antibody (ab2609)
    Immunoprecipitation - Anti-eIF4G1 antibody (ab2609)
    All lanes : Anti-eIF4G1 antibody (ab2609) at 1 µg/ml

    Lane 1 : HeLa cells immunoprecipitated by ab2609 at 6 µg per reaction
    Lane 2 : HeLa cells immunoprecipitated by control IgG at 6 µg per reaction

    Exposure time: 10 seconds
  • Western blot - Anti-eIF4G1 antibody (ab2609)
    Western blot - Anti-eIF4G1 antibody (ab2609)

    50µg (lane 1) and 150µg (lane 2) rat liver lysate, separated on 7.5% acrylamide SDS-PAGE gel. Detected using ab2609 at 1:1000 (A), 1:5000 (B) and 1:10000 (C) dilution by ECL. 

    50µg (lane 1) and 150µg (lane 2) rat liver lysate, separated on 7.5% acrylamide SDS-PAGE gel. Detected using ab2609 at 1:1000 (A), 1:5000 (B) and 1:10000 (C) dilution by ECL.
  • Immunocytochemistry/ Immunofluorescence - Anti-eIF4G1 antibody (ab2609)
    Immunocytochemistry/ Immunofluorescence - Anti-eIF4G1 antibody (ab2609)
    ICC/IF image of ab2609 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2609, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Wu H  et al. N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription. Viruses 8:N/A (2016). Read more (PubMed: 27447663) »
  • Van Rechem C  et al. Lysine demethylase KDM4A associates with translation machinery and regulates protein synthesis. Cancer Discov 5:255-63 (2015). WB ; Human . Read more (PubMed: 25564516) »
See all 16 Publications for this product

Publishing research using ab2609? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 13 Abreviews or Q&A

Immunoprecipitation abreview for Anti-eIF4G1 antibody

 Poor
Abreviews
abreview image
Application
Immunoprecipitation
Sample
Mouse Cell lysate - whole cell (mouse embryonic stem cells (J1))
Total protein in input
600000 cells
Immuno-precipitation step
Protein G
Specification
mouse embryonic stem cells (J1)
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Abcam user community

Verified customer

Submitted Aug 14 2018

Western blot abreview for Anti-eIF4G1 antibody

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Mouse Cell lysate - whole cell (mouse embryonic stem cells (J1))
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
mouse embryonic stem cells (J1)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Abcam user community

Verified customer

Submitted Mar 02 2018

Western blot abreview for Anti-eIF4G1 antibody

 Excellent
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Nasal epithelial cells, bronchial epithelial cells)
Gel Running Conditions
Non-reduced Denaturing (6%)
Loading amount
20 µg
Specification
Nasal epithelial cells, bronchial epithelial cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Dr. Kyeongah Kim

Verified customer

Submitted Jun 16 2017

Question

We recently bought eIF4G1 (ab2609). In the product info sheet you state that the protein is more susceptible to degradation than other proteins - can you tell me the source of this observation e.g primary references etc. We work with brain lysates and have problems with the antibody. I would be grateful for a prompt reply as we are under pressure from a competitor. Thanks,

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Answer

Thank you for your enquiry. The information on the datasheet was from a mail correspondence with the product originator back in June 2004 (please see FAQ section on datasheet). I am afraid that with our current mail system, we can not retrieve any old enquiries before the year 2006 therefore I am not able to provide more information on the enquiry itself. However, I did manage to do an internet search and found the following publication that mentions the susceptibility of eIF4G1 degradation. Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis. FEBS Letters. Volume 451, Issue 3, 28 May 1999, Pages 332-336 http://linkinghub.elsevier.com/retrieve/pii/S0014579399006146 I am afraid that I can't provide any more information that already stated above but I hope that the publication and the FAQ information will be helpful.

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Question

I used this antibody on mouse brain extract and it works. It recognizes the expected protein around 176 KDa but other bands under this molecular weight are also present, it is likely due to the degradation of eIF4G or to an aspecific cross-reacts.

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Answer

Thank you for your enquiry. EIF4G is more susceptible to degradation compared to other proteins, especially from some tissue sources such as liver. This is true even when tissue is stored at frozen. SDS-PAGE sample buffer may improve the stability, but samples that are stored frozen may show degradation bands that have been described in the literature. Therefore if multiple bands are observed in WB, it is likely due to the degradation of eIF4G rather than non-specificity of the antibody. I would therefore recommend to take all precautions possible to prevent degradation of eIF4G (fresh protease inhibitors, keeping samples on ice at all times, using SDS-PAGE loading buffer), Please do not hesitate to contact us if you require further assistance,

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Question

BATCH NUMBER 53909 ORDER NUMBER seenotes DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Total cell extract from HELA, HCT116 or MEFs. OR precipitated material from CAP-binding assay. PRIMARY ANTIBODY Abcam/ Ab2609-50/ PBS, 2%BSA and 0.1%tween-20. 2 hrs at room temperature. Wash 3 times 5 minutes with PBS 0.1% tween-20. SECONDARY ANTIBODY BD Pharmingen, 1:20,000 in PBS 0.1% tween-20, 1 hr at room temperature, wash 3 times 5 minutes with PBS 0.1% tween-20. DETECTION METHOD ECL plus POSITIVE AND NEGATIVE CONTROLS USED Positive controls from previous experiments used. With our previous batch of antibody these used to worked perfectly! ANTIBODY STORAGE CONDITIONS 4 degrees SAMPLE PREPARATION 50mM Hepes-KOH pH 7.5,150mM KCl,1mM EDTA pH 8,2mM DTT,0.2 %Tween-20 + complete protease inhibitor (Roche). AMOUNT OF PROTEIN LOADED 40 microgram ELECTROPHORESIS/GEL CONDITIONS Non-reducing gradient gel 6-20% TRANSFER AND BLOCKING CONDITIONS Transfer buffer from Biorad supplemented with 200 mL methanol per 1 L buffer. Transfer at 100V and 4 degrees. Blocking overnight in PBS+ 5%non fat milk gentle aggitation. Transfer tested by Ponceau Red staining. 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? We tried several different things to optimize the detection, however unsuccessfully. Overnight incubation with antibody, different membrane (PVDF), different dilution of primairy (from 1:1000-1:4000), different wash periods, very long exposures. NOTHING worked! ADDITIONAL NOTES Order# 4500027150. We were always very pleased with the performance of this antibody. It gave good strong signals. However with this new batch that we started to use approximately 3 weeks ago, nothing works. Even after extensive optimization! Are there any known problems with this particular batch (53909)? We really urgently need to perform some blots!

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Answer

I'm very sorry to hear you are having problems with a new vial of ab2609. Thank you for taking the time to fill in the questionnaire, it is very useful for me to understand your problem. I have checked the batches you have used and found out that they are actually derived from the same batch of the originator. Therefore it is likely that there was a problem with your particular vial, maybe it was damaged during transport or storage. I would like to send you a replacement vial immediately, could you please give me the Abcam order number or PO number and the name of the person who placed the order. I look forward to hearing from you,

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Question

ANTIBODY CODE ab2609 BATCH NUMBER 64851 ORDER NUMBER 25164797 DESCRIPTION OF THE PROBLEM Multiple bands. Should produce bands between 200-220 kDa. Instead of the 220 kDa band, produces non specific multiple bands. The western blot showed two strong bands of aprox 70 and 40 kDa, in addition to the multiple bands. It doesn't work in immunoprecipitation neither. We want a refund for this product. Our order No is 251647979. SAMPLE Baby Hamster Kidney cells (BHK-21 cells) PRIMARY ANTIBODY anti-eIF4G1 (ab2609) diluted 1/1000 in 5% BSA in PBS-Tween. Incubated overnigth at 4C with gentle agitation. Washed 3 times in PBS-Tween SECONDARY ANTIBODY anti-rabitt/HRP (Cell Signaling Technology) diluted 1/2000 in 5% BSA in PBS-Tween. Washed 4 times in PBS-Tween. We reprobed the membrane with eIF4E antibody from Cell Signaling Technology, using the same conditions and we detected a single 25 kDa band corresponding with eIF4E. So it seems, that there is not problem with our western blot conditions or our samples. We wish to send you pictures of the western blots. DETECTION METHOD ECL (Amersham) POSITIVE AND NEGATIVE CONTROLS USED The same samples were blotted using the following antibodies: eIF4E (Cell Signaling Technology), using the same secondary antibody. Also we used Mab 10E10 anti-PABP and Mab 1119 anti-ICP27 antibodies(one cell extract was infected with HSV). These antibodies performed as we expected, so the samples and western blott procedures are ok. ANTIBODY STORAGE CONDITIONS 4 C SAMPLE PREPARATION Monolayer of BHK-21 cells was washed three times in PBS and resuspended in HEPES extract buffer (50 mM HEPES pH 7.5, 50 mM NaCl, 0.1 % Nonidet P40, plus protease inhibitor tablets, Roche). Cells were sonicated and cellular debris pelleted. Cell extracts were kept at -70C and protein measured by Bradford (BIO-RAD protein assay). All procedures on ice. AMOUNT OF PROTEIN LOADED 20 micrograms ELECTROPHORESIS/GEL CONDITIONS 10 % SDS-PAGE TRANSFER AND BLOCKING CONDITIONS Submerged electrophoretic transfer (25 mM Tris, 192 mM glycine, 20% methanol) for 1.5 h at 250 mA. Blocking in 5% w/v nonfat dry milk in PBS-Tween HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? same conditions

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Answer

As requested we will refund you for the cost of this antibody. I note from our datasheet that this antibody has only been tested for cross reactivity with human and rat and you advise that you were using Baby Hamster Kidney cells. However, the fact that other antibodies to eIF4G1 have worked successfully in your research means that there could also be a problem with the vial you received or with the quality of the product. Having checked other enquiries, I see there is another unfavourable report from a customer using this antibody that we received on Monday 28-June-2004 - please see the enquiries section of the online datasheet for further details. I will ask the originator of the antibody to re-test this product and if I am not satisfied with the result will remove it from our range. Our reputation is far too valuable to risk selling products that do not work as we say they should. Thank you for your feedback, and once again apologies for the inconvenience caused. [10 September 2004] We have confirmed the quality of this product with the originator. The problems this researcher experienced are most likely to have arisen because of the break down of eIF4G1 which is very unstable rather than problems with the quality of this antibody. eIF4G breaks down very quickly and it needs to be the first thing probed for when the test material comes out of the freezer. The shorter the time between Western blot and freeze clamping the tissues or freezing the cells the better. It is also work noting that I had misread the original enquiry from this researcher. The other primary antibodies used were raised against different proteins which would be more stable that eIF4G. We are confident that this antibody should remain in our range and would welcome feedback from other researchers using it.

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Question

Also, since we confirmed with the originator that this antibody should be able to recognize both 4G I and II, could ask to him/her whether the two bands we detect could be the two isoforms. If my samples were suffering from degradation I think I should have detected multiple bands, whereas I detect two distinct bands one running with the 250 kDa and the other with the 100 kDa. Thanks a lot again, As of today I have completed five westerns and two IP reactions using the ab2609. There is absolutely no match between the band patterns in whole lysate and the IP samples run on the same gel where a rat liver lysate and a rat colon carcinoma cell line sample were included as controls. Therefore, we have decided to stop wasting our reagents and time using the ab2609 in our experiments. I would like to send the vial (there is still 35-40 micrograms left) back to you if you could give me the mailing information. In the meantime our PI is trying to come up with an alternative antibody which we could get for the credit of the ab 2609. I will let you know when she gives me that information. I would like to thank to you specially for assisting our problems and trying to help us in our assays.

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Answer

Thank you for your patience. I'm sorry to hear that you are experiencing trouble with this antibody. I'm trying to obtain more information from the originator to help figure just what may be going on, but am still waiting to hear back. There is no need to send back the remaining antibody. I will send you another antibody as a replacement, or give you a refund. Can you clarify what you mean by there being no match between your IP samples and the controls? What size band did you see with the rat liver lysate?

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Question

I have just completed a western of a cap-binding assay using the ab2609. In the whole cell lysates I detected two bands one runs with the 250 kDa, the other runs with the 100 kDa. The cap-bound fractions just show the 250 Kda band but not the 100 kDa band. Am I looking at eIF4G now, or some other non-specific band. These were rat lysates and 10 micrograms were loaded on the gel. Since I have not heard from you about the buffer for IP I will use a published buffer formulation for my huge experiment. Please keep me posted, because I really have to make sure that these bands are not non-specific. Thank you very much indeed,

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Answer

Thank you for your patience. The originator of ab2609 has provided me with the following information: "eIF-4G migrates in SDS-PAGE just above myosin standard. If colored molecular weight markers are used it can be just above or just below dyed myosin standards depending on the manufacturer. EIF4G is more susceptible to degradation compared to other proteins, especially from some tissue sources such as liver. This is true even when tissue is stored at frozen. SDS-PAGE sample buffer may improve the stability, but samples that are stored frozen may show degradation bands that have been described in the literature." I don't know if you have done this already, but it is recommended to use rat liver lysate as a positive control. I have not received information regarding the IP buffers, but the originator has said that those in the literature should work fine. If you have any more questions or concerns, please do let me know.

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Question

Does ab2609 detect both isoforms of eIF4G1? Do exons 10 and 11 lie in the region where there is homology between the two isoforms?

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Answer

The peptide used to generate the antibody is common to all 5 isoforms shown for SwissProt entry Q04637 (http://au.expasy.org/cgi-bin/niceprot.pl?Q04637). It lies between residues 560 and 660 of isoform A as defined in SwissProt entry Q04637.

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1-10 of 13 Abreviews or Q&A

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