Overview

  • Product name
    Anti-GABA A Receptor alpha 5 antibody
    See all GABA A Receptor alpha 5 primary antibodies
  • Description
    Rabbit polyclonal to GABA A Receptor alpha 5
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ELISA, WB, IHC-Fr, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Rabbit, Horse, Chicken, Guinea pig, Cow, Cat, Dog
  • Immunogen

    Synthetic peptide within Mouse GABA A Receptor alpha 5 aa 180-229 (internal sequence). The exact sequence is proprietary. Numbering is based off mature sequence
    Sequence:

    WTNGSTKSVVVAEDGSRLNQYHLMGQTVGTENISTSTGEYTIMTAHFHLK


    Database link: Q8BHJ7
    (Peptide available as ab137922)

  • Positive control
    • Skeletal Muscle (Mouse) Whole Cell Lysate (ab3869) can be used as a positive control in WB. Mouse muscle lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab10098 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 Use at an assay dependent concentration. Positive result against immunising peptide.
WB 1/1000. Detects a band of approximately 52 kDa (predicted molecular weight: 55.4 kDa).Can be blocked with GABA A Receptor alpha 5 peptide (ab137922). Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function
    GABA, the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel.
  • Sequence similarities
    Belongs to the ligand-gated ion channel (TC 1.A.9) family. Gamma-aminobutyric acid receptor (TC 1.A.9.5) subfamily. GABRA5 sub-subfamily.
  • Cellular localization
    Cell junction > synapse > postsynaptic cell membrane. Cell membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • GAA 5 antibody
    • GAA5 antibody
    • GABA(A) receptor subunit alpha-5 antibody
    • GABRA 5 antibody
    • Gabra5 antibody
    • Gamma aminobutyric acid GABA A receptor alpha 5 antibody
    • Gamma aminobutyric acid GABA A receptor alpha 5 precursor antibody
    • Gamma aminobutyric acid receptor alpha 5 subunit precursor GABA A receptor antibody
    • Gamma-aminobutyric acid receptor subunit alpha-5 antibody
    • GBRA5_HUMAN antibody
    • GC138184 antibody
    see all

Images

  • Predicted band size: 55.4 kDa
    Observed band size: 52 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 40 kDa (possible non-specific binding)

  • All lanes : Anti-GABA A Receptor alpha 5 antibody (ab10098) at 1/1000 dilution

    All lanes : Mouse brain lysate

    Lysates/proteins at 40 µg per lane.

    Secondary
    All lanes : IRDye® 680LT-conjugated donkey anti-rabbit IgG polyclonal (undiluted)

    Performed under reducing conditions.

    Predicted band size: 55.4 kDa
    Observed band size: 53 kDa why is the actual band size different from the predicted?


    Exposure time: 6 minutes

    See Abreview

  • ICC/IF image of ab10098 stained SHSY5Y cells. The cells were 4% formaldehyde 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 (ab10098, 5µg/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.

  • ab10098 staining GABA A Receptor alpha 5 in rat hippocampus sections by Immunohistochemistry (Frozen sections).
    Rats were transcardially perfused with 4% PFA, the brain was cryoprotected in 30% sucrose before flash freezing. Coronal sections were taken using cryostat (20 micron sections) and mounted onto slides. Tissue was fixed in paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were then blocked using 10% serum for 1 hour at 20°C and then incubated with ab10098 at a 1/200 dilution for 16 hours at 4°C. The secondary used was an Alexa Fluor® 594 goat anti-rabbit IgG (H+L) used at a 1/600 dilution.

    An antigen retrieval step improved results (5% SDS for 5 minutes before standard IHC protocol).

    See Abreview

  • Anti-GABA A Receptor alpha 5 antibody (ab10098) at 1 µg/ml + Mouse Liver Lysate

    Predicted band size: 55.4 kDa

References

This product has been referenced in:
  • Hu D  et al. Bumetanide treatment during early development rescues maternal separation-induced susceptibility to stress. Sci Rep 7:11878 (2017). Read more (PubMed: 28928398) »
  • Modi KK  et al. Cinnamon Converts Poor Learning Mice to Good Learners: Implications for Memory Improvement. J Neuroimmune Pharmacol 11:693-707 (2016). Read more (PubMed: 27342118) »
See all 8 Publications for this product

Customer reviews and Q&As

1-10 of 23 Abreviews or Q&A

Answer

Thank you for providing me with that information. This will be very helpful for us in trying to understand what may have contributed to the problems observed.

I would like to point out with the new antibody (ab83003). This antibody has shown good resultsthe membrane wasblocked with 5% non-fat dry milk in 0.05% PBS-T. I would therefore try this if possible. Also, as mentioned in my previous email, we have not tested this antibody with mouse samples, but due to the sequence homology with the immunogen used, we would expect it to react (although we cannot guarantee it). It would be worthwhile using the mouse positive control lysate that my colleague provided for you to try with the antibody.

I would also suggest, that it may be worthwhile to try the ab10098 if you have time again with a different blocking conditions. I would suggest either 5% milk in PBST with 1% milk in the antibody diluent, or 3% BSA in PBST, again with 1% BSA in the antibody diluent buffer.

I hope this information has been of help. Please do let me know how the new antibody performs in your experiments and if you continue to have any problems.

I look forward to hearing how you get on. Until then, I wish you all the best with your research.

Read More
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Rat Tissue sections (Brain)
Antigen retrieval step
None
Permeabilization
No
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Fixative
Paraformaldehyde

Mrs. Olga Cravetchi

Verified customer

Submitted Jul 12 2016

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing (8%)
Sample
Mouse Cell lysate - other (Brain)
Specification
Brain
Blocking step
LI-COR Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jan 22 2015

Question
Answer

Thank you for contacting us regarding controls to use with ab10098.

Use of a blocking peptide will determine whether non-specific binding of an antibody to proteins other than the antigen is occurring.I have been able to add the blocking peptide to this product to our catalog. The peptide,GABA A Receptor alpha 5 peptide (ab137922) may be found at the following link:

https://www.abcam.com/GABA-A-Receptor-alpha-5-peptide-ab137922.html


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Answer

Thank you for calling us today to report the problems you are continuing to have with the anti-GABA A Receptor alpha 5 antibody (ab10098). As discussed over the phone, I have arranged for a replacement vial of the antibody ab83003 to be sent to you. This should be with you next week.

Please be aware that as we have not tested it with mouse samples, I would not be able to guarantee that is will work with this tissue type. As the immunogen sequence matches with 100% homology to themouse protein we would predict that it would work.

It has however been confirmed to detect the human protein in Western blotting as you are hoping to perform.

Please do let me know how you get on with the new antibody.

Also, as discussed over the phone, I'd like to find out a little more about how the antibody ab10098 has been used. To this end, I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. This information will also allow us to investigate this case internally and initiate additional testing where necessary.

I look forward to receiving your reply.

Read More

Answer

Thank you for contacting us and your interest in our products.
We would indeed be very interested in your customer trying anti-GABA A Receptor alpha 5 antibody (ab10098) with rat samples. By comparing the sequence of the immunogen used against the sequence of the rat protein, a homology of 100% is observed. We would therefore expect the antibody to be able to detect the rat protein.
I have therefore issued the following discount code:


More information on the testing discount scheme can be found from the following link:
www.abcam.com/collaborationdiscount
I hope this information has been of help. If you require any further information please do not hesitate to contact us again.

Read More

Answer

Thank you for contacting us.


I now have some more information to share with you about the specificity of Anti-GABA A Receptor alpha 5 antibody (ab10098). A western blot with mouse muscle tissue lysate detecting a clean band at theexpectedsized of 52 kDa has been performed.The antibody has also been used to stain SHSY5Y cells as well as rat hippocampus sections (please refer to datasheet of ab10098) with the expected results.


Unfortunately, additional specificity tests, for example, a western blot in the presence of the blocking peptide or testing of knock out models have not been performed with this antibody to our knowledge.


In performing a blast using the immunogen used to raise the antibody (region between amino acids 231-245 of Human GABA A Receptor alpha 5), no protein is similar to the sequence (none above 73%) apart from the GABA A Receptor alpha 5 (SwissProt reference http://www.uniprot.org/uniprot/P31644). We would therefore not expect the antibody to cross react with other proteins, however, as this has not been conclusively tested we cannot guarantee that it would not.


I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

Read More

Answer

Thank you for your reply.

Based on all of the information you have provided, I am wondering if the problem is due to auto fluorescence, as I cannot think of any other way that you would see background signal in your no secondary condition, as there should be no fluorescent signal present in your sample, since you havent added any.


Try incubating the sections in a solution of 1% Sodium Borohydride (NaBH4)in 0.1M Phosphate buffer (not PBS)
for about 30 minutes. This substance will quench autofluorescnce from free aldehyde groups left over from fixation
and should resolve the issue.

Please let me know if there is anything else that I can help you with.

Read More

Answer

Thank you for your reply.

Is your negative control, the sections without any primary antibody present? I ask as I am trying to narrow down if the issue is with the primary or secondary antibody.

In the original protocol information you sent to me, you mentioned that you had altered antibody concentrations, which concentrations did you try (for both primary and secondary) and did altering these concentrations make any difference to the level of background seen.

Also switching to a serum block rather than BSA block may prove to be very helpful, especially if the secondary antibody you are using was raised in goat.

I appreciate your patience in this matter, while I try and collect all of the relevant information.

Read More

Answer

Thank you for contacting Abcam.

I would be happy to help you with some support regarding ab10098. Could you please answer the questions below about the protocol that you are using and also include the type of tissue/cells you are using and the species that it comes from:

1) Fixation step

Fixation time

Fixation temperature

2) Antigen retrieval method (time and procedure)

3) Permeabilization method:

Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?

Permeabilizing agent and concentration:

4) Blocking agent (eg BSA, serum…):

Concentration

Blocking time

Blocking temperature

5) Endogenous peroxidases blocked?

Endogenous biotins blocked?

6) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution

Diluent buffer

Incubation time

7) Secondary antibody:

Species:

Reacts against:

Concentration or dilution

Diluent buffer

Incubation time

Fluorochrome or enzyme conjugate

8) Washing after primary and secondary antibodies:

Buffer

Number of washes

9) Detection method

10) How many times have you run this staining?

Do you obtain the same results every time?

What steps have you altered to try and optimize the use of this antibody?

Read More

1-10 of 23 Abreviews or Q&A

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