Overview

  • Product name
    Anti-HA tag antibody [4C12]
    See all HA tag primary antibodies
  • Description
    Mouse monoclonal [4C12] to HA tag
  • Host species
    Mouse
  • Specificity
    The antibody detects HA-tag fused to the C-terminus or N-terminus of targeted proteins in transfected cells. The antibody works in ChIP but less well than ab9110 (see ChIP data).
  • Tested applications
    Suitable for: ELISA, ICC/IF, ChIP, IP, WBmore details
  • Species reactivity
    Reacts with: Species independent
  • Positive control
    • WB: Rab6-HA transfected HEK-293T whole cell lysate. IP: Rab6-HA transfected HEK-293T whole cell lysate. ICC/IF: Baby hamster kidney cell expressing HA-tag CFTR protein. ChIP: Xenopus laevis oocytes injected with mRNA for HA-tagged human BORIS.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    PBS pH7.2 with 50% glycerol, 1% BSA and 0.02% sodium azide
  • Concentration information loading...
  • Clonality
    Monoclonal
  • Clone number
    4C12
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab1424 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
ELISA 1/1000. PubMed: 17167031
ICC/IF Use a concentration of 10 µg/ml.
ChIP Use a concentration of 3 µg/ml. Use 3 µg per 25 µg chromatin (see figure legend).
IP Use a concentration of 10 µg/ml.
WB Use a concentration of 1 µg/ml.

Target

  • Relevance
    Human influenza hemagglutinin (HA) is a surface glycoprotein required for the infectivity of the human virus. The HA tag is derived from the HA molecule corresponding to amino acids 98-106 has been extensively used as a general epitope tag in expression vectors. Many recombinant proteins have been engineered to express the HA tag, which does not appear to interfere with the bioactivity or the biodistribution of the recombinant protein. This tag facilitates the detection, isolation, and purification of the proteins.
  • Alternative names
    • HA epitope tag antibody
    • HA1 antibody
    • HA2 antibody
    • hemagglutinin antibody
    • Hemagglutinin HA1 chain antibody
    • Hemagglutinin HA2 chain antibody
    see all

Images

  • Anti-HA tag antibody [4C12] (ab1424) at 1/1000 dilution + HEK293T whole cell lysate over-expressing HA-tagged Rab6 at 20 µg

    Secondary
    HRP-conjugated goat anti-mouse polyclonal IgG at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 5 seconds


    Blocked with 5% milk for 30 min at 25°C

    See Abreview

  • This picture shows baby hamster kidney cell expressing HA-tag CFTR protein. This type of cells were grown on coverslip and immunofluorescence was performed. Dilution of 1:200 was used and incubated for 2hrs with antibody.

  • Recombinant HA-tagged Rab6 was immunoprecipitated from 1 mg transfected HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell extract with 2 μL ab1424. 

    Lane 1: Rab6-HA transfected HEK-293T whole cell extract (Input)

    Lane 2: ab1424 IP in Rab6-HA transfected HEK-293T whole cell extract.

     

  • Xenopus laevis oocytes were injected with mRNA for HA-tagged human BORIS. Chromatin was prepared according to the Abcam X-ChIP protocol. Oocytes were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 3µg of ab1424 (anti-HA, light blue)  and 3µg of ab18337 (anti-BORIS, dark blue), and 20µl of Protein A/G sepharose beads. A non-specific antibody was used as a control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach).

References

This product has been referenced in:
  • Peng J  et al. Drosophila Fezf coordinates laminar-specific connectivity through cell-intrinsic and cell-extrinsic mechanisms. Elife 7:N/A (2018). Read more (PubMed: 29513217) »
  • Chang YC  et al. Temporal and spatial order of photoreceptor and glia projections into optic lobe in Drosophila. Sci Rep 8:12669 (2018). Read more (PubMed: 30140062) »
See all 25 Publications for this product

Customer reviews and Q&As

1-10 of 13 Q&A

Answer

Thank you for your reply.

You can find out more about the 100% Abreview Testing Discount Program below:

https://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount



For all the antibodies that you are interested in, we already guarantee them to work in ChIP experiments, regardless of species. Therefore, they would not qualify for the testing discount program, but they are covered under our Abpromise.

If there is anything else I can help you with, or if you are interested in any of our other antibodies that have not been tested in ChIP, but you might be interested in them, then please let me know.

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Answer

Thank you for contacting Abcam. Because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

If the product is to be used in an untested species or application, you may be eligible for our testing discount program if the antibody has not yet been purchased. Please contact our Scientific Support team by replying to this email prior to purchase for more information.

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

https://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

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Answer

Thank you for contacting us. Both antibodies have not yet been tested in any IHC application (neither IHC-P or IHC-Fr). We do have a testing discount program that you can use in that case. Here is some information about how the testing discount program works: The testing discount program is for customers who like to use an antibody/protein on an untested species/application. You would purchase the antibody at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody at the full price (100%) of the antibody you have tested. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount. I'd be happy to issue you a discount code for one or both antibodies. Please let me know. Also, please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

Thank you for your enquiry. The immunogen was YPYDVPDYAC. Please let me know if you have any more questions.

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Answer

Thank you for your enquiry. Jen is out of the office, so I am answering her correspondence. I understand that you used a third antibody successfully in this protocol, but each antibody is different and sometimes must be optimized to work well. What is the specific problem you are getting with each antibody? Are you seeing no signal whatsoever, high background, etc? What blocking solution did you use? 5% milk? BSA? How long and at what temperature did you incubate with the primary antibody? The protocol you included said 2-3 hours at room temperature or overnight at 4 degrees. 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 your phone call and I'm sorry to hear that you are experiencing difficulty with this antibody. We have not received any other complaints regarding this batch and so it's a possibility that this latest vial which you received went off at some point during the shipping process. I have arranged for a replacement vial to be sent to you free of charge. This is on order# 87816 and you should receive this Wednesday. Please let me know how the replacement works out for you and if you have any additional questions.

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Question

Here are some answers to your questions: 1. Please describe the problem (high background, wrong band size, more bands, no band etc).---Non specific bands all over the blot 2. On what material are you testing the antibody in WB? Species? Cell extract/ Nuclear extract?--- cell lysates from BHK cells Purified protein? Recombinant protein? 3. How much protein did you load?--Loaded around 10ul of lysate, did not quantify. How did you prepare the lysate for the analysis (protease inhibitors etc)?---Lysates were prepared using 1%DM (dodecy maltoside) with protease inhibitor cocktail. Did you heat the samples?--No, its a membrane protein, and heating would disrupt it 4. Primary Antibody Specification (in which species was it raised against)?--Mouse HA tag (Abcam) At what dilution(s) have you tested this antibody?--1:1000, 1:10,000 Incubation time, wash step?:1hr followed by 3 washes for 5min each 5. Secondary Antibody Specification (in which species was it raised against)?--goat anti mouse IR labbled At what dilution(s) have you tested this antibody?: 1:5000 Incubation time, wash step?--45min followed by 3 washes for 5min each Do you know whether the problems you are experiencing come from the secondary?---its not a problem of secondary, all my blots are fine under similar conditions with same secondary 6. What detection method are you using?--Odyssey scanner bwith detects IR labelling 7. Background bands Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a ?No primary? control)---yes Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4?C with agitation)---the odyssey blocking buffer is recommended for 1hr at 37degC Are your washing steps sufficiently stringent? ---we use 3 five minute washes after primary and secondary At what size are the bands migrating? Could they be degradation products of your target? --they all are non-specific Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts How many times have you tried the Western?--4X Do you obtain the same results every time e.g. are background bands always in the same place? yes What steps have you altered?--different antibody dilutions 9. Did you apply positive and negative controls along with the samples? Please specify.--no

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Answer

Thank you for providing me with details regarding your protocol and also for the image you submitted. We have had some good feedback regarding this antibody in Western blotting, and customers have reported seeing a clean signal using ab1424 at a dilution of 1:1000. Since the extra bands that you are seeing are not due to your secondary, and I don't think diluting the primary any further is going to help, I can offer you a replacement vial of this antibody to try. What was the lot number that you received (it is located on the vial)? Also, what was the Abcam order number or purchase order number that was used for your order for ab1424? Thanks again, and I look forward to hearing from you.

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Answer

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab1424. Could we get a detailed protocol from you, please? We have some general questions, the answers to which will enable us to investigate this matter as quickly as possible. Also, please include the lot number that you received (it is located on the vial), and the Abcam order number or purchase order number that was used. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? 6. What detection method are you using? 7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? 9. Did you apply positive and negative controls along with the samples? Please specify.

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Answer

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with this antibody. Without knowing more details regarding your protocol, I would first suggest decreasing the concentrations of the primary and secondary antibodies and also run a secondary-only control (no primary antibody) to ensure that the non-specific bands are not due to your secondary antibody. If you continue to have trouble with ab1424, please let me know and also please answer the questions below regarding your protocol. Also, please include the lot number that you received (located on the vial) and the Abcam order number or purchase order number that was used. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? 6. What detection method are you using? 7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? 9. Did you apply positive and negative controls along with the samples? Please specify.

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Question

I am sending you the answers I got from my customer in the attached file "Abcam Answer" and the image of the HA-blot. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). - high background and more bands (we suspect a sort of cross reaction) > 2. On what material are you testing the antibody in WB? > .Species? > .Cell extract/ Nuclear extract? - Cell extracts by direct lysis and nuclear extracts with sucrose buffer and Ab buffer (Luscher B. et al, Molecular and Cell Biology, June 1988, p. 2504-2512) > .Purified protein? > .Recombinant protein? > > 3. How much protein did you load? 50? and 100? > .How did you prepare the lysate for the analysis (protease inhibitors etc)? See above (We used protease inhibitors Roche) > .Did you heat the samples? We boiled samples 10’ before loading > > 4. Primary Antibody > .Specification (in which species was it raised against)? Mouse > .At what dilution(s) have you tested this antibody? 1?/? > .Incubation time, wash step? 1 hour and after twice wash 8’ each > > 5. Secondary Antibody > .Specification (in which species was it raised against)? Mouse > .At what dilution(s) have you tested this antibody? 1:5000 > .Incubation time, wash step? 3X wash 10’ each > .Do you know whether the problems you are experiencing come from the secondary? In my experience no problems come from the secondary antibody > > 6. What detection method are you using? ECL (Amersham kit) > > 7. Background bands > .Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a "No primary" control) We didn’t use a “No primary” control but we never had problems with secondary antibody in others blot > .Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) Yes > .Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) Yes > .At what size are the bands migrating? Could they be degradation products of your target? About 75KD (Usually we load a control samples lysate obtain from cells with empty vector, thus without HA. In this case also in this control lane we can see aspecific band.) > .Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) > > 8. Optimization attempts > .How many times have you tried the Western? 3 times > .Do you obtain the same results every time e.g. are background bands always in the same place? Yes > .What steps have you altered? Increased blocking time and decreased time in secondary antibody incubation > > 9. Did you apply positive and negative controls along with the samples? Please specify. We didn’t load positive controls but we had negative controls (see above)

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Answer

Thank you for your customer's details regarding their protocol. At this point I would recommend decreasing the concentration of the primary antibody and to also run a secondary antibody control (no primary) to ensure that this cross-reactivity is not coming from the secondary. Let me know how this turns out for you customer...thanks.

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1-10 of 13 Q&A

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