Overview

  • Product name

    Anti-gamma Sarcoglycan antibody
    See all gamma Sarcoglycan primary antibodies
  • Description

    Rabbit polyclonal to gamma Sarcoglycan
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Full length Human protein (NP_000222.1)

  • Positive control

    • Human kidney tissue lysate, Transfected 293T cell line IHC-P: FFPE human heart tissue sections.

Properties

Applications

Our Abpromise guarantee covers the use of ab104478 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 Use a concentration of 1 µg/ml. Predicted molecular weight: 32 kDa.
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.

Target

  • Function

    Component of the sarcoglycan complex, a subcomplex of the dystrophin-glycoprotein complex which forms a link between the F-actin cytoskeleton and the extracellular matrix.
  • Tissue specificity

    Expressed in skeletal and heart muscle.
  • Involvement in disease

    Defects in SGCG are the cause of limb-girdle muscular dystrophy type 2C (LGMD2C) [MIM:253700]. LGMD2C is characterized by progressive muscle wasting from early childhood.
  • Sequence similarities

    Belongs to the sarcoglycan beta/delta/gamma/zeta family.
  • Cellular localization

    Cell membrane > sarcolemma. Cytoplasm > cytoskeleton.
  • Information by UniProt
  • Database links

  • Alternative names

    • 35 kDa dystrophin associated glycoprotein antibody
    • 35 kDa dystrophin-associated glycoprotein antibody
    • 35DAG antibody
    • 35kD dystrophin associated glycoprotein antibody
    • 35kDa dystrophin-associated glycoprotein antibody
    • A4 antibody
    • DAGA4 antibody
    • DMDA antibody
    • DMDA1 antibody
    • Gamma SG antibody
    • Gamma-sarcoglycan antibody
    • Gamma-SG antibody
    • LGMD2C antibody
    • MAM antibody
    • MGC130048 antibody
    • Sarcoglycan gamma antibody
    • SCARMD2 antibody
    • SCG3 antibody
    • SGCG antibody
    • SGCG_HUMAN antibody
    • TYPE antibody
    see all

Images

  • IHC image of gamma Sarcoglycan staining in human heart formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. 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 ab104478, 10µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer 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.

  • Anti-gamma Sarcoglycan antibody (ab104478) at 1 µg/ml + Human kidney tissue lysate at 50 µg

    Predicted band size: 32 kDa

  • All lanes : Anti-gamma Sarcoglycan antibody (ab104478) at 1 µg/ml

    Lane 1 : gamma Sarcoglycan transfected 293T cell lysate
    Lane 2 : Non-transfected 293T cell lysate

    Lysates/proteins at 25 µg per lane.

    Predicted band size: 32 kDa

References

This product has been referenced in:

  • van den Bergen JC  et al. Clinical characterisation of Becker muscular dystrophy patients predicts favourable outcome in exon-skipping therapy. J Neurol Neurosurg Psychiatry N/A:N/A (2013). Human . Read more (PubMed: 23667215) »
See 1 Publication for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Application
Immunohistochemistry free floating
Sample
Rabbit Tissue sections (Olfactory bulb)
Specification
Olfactory bulb

Abcam user community

Verified customer

Submitted Jan 05 2017

Answer

Thank you for your enquiry.

The sarcoglycan protein is located in the cell membrane and expressed in kidney, so the membrane kidney lyaste ab30193 should be suitable for both these sarcoglycan antibodies. However, they may like to consider a whole cell lysate instead, or in addition:

ab30203Kidney (Human) Tissue Lysate - adult normal tissue
https://www.abcam.com/index.html?datasheet=30203 (or use the following: https://www.abcam.com/index.html?datasheet=30203).

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Question

I have received some more information in this matter. Please check.


I have used the same protocol for other sarcoglycan like Alpha and Delta it has worked well.But i dint get any result with this particular proteins Beta and Gamma (abcam).

Extraction is good enough, loading control is good enough.

Protease inhibitor is not required here because as soon as i minice the tissue i add stringent extraction buffer which has high amount of SDS,where protease usually donot act. Extraction procedure has DDT also.

Yes secondary antibody is working.



I cannot estimate the protein because extraction buffer has CCB in it. But i feel i have added enough protein (other sarcoglycan has worked)



I even want to note that in case of Beta sarcoglycan ab55683 C2C12 cell line has band corr to 50kDa nothttp://www.rediffmail.com/cgi-bin/red.cgi?account_type=1&red=http://43kDa.So&isImage=0&BlockImage=0&rediffng=0i feel the band what i could get in C2C12 was a non specific band.waiting forward for your reply.










I have used normal human skeletal muscle biopsies as positive control.we dont have acess to other positive controls as suggested in product sheet. I have used the same human skeletal muscle biopsies for other sarcoglycan like Alpha and delta ,which has worked before with the same blotting technique.But dint work for the following antibody ab104478 (no bands in either of the sample neither in C2C12 nor in Skeletal muscle sample) and ab55683 (band in C2C12 and no band in skeletal muscle sample).

Regards

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Answer

Thank you for your response and for sending some additional information.
After reading through the detailed protocol you forwarded to Abcam, I would like to make the following comments/suggestions. Please would you be so kind to ask your troubled customer to read this message carefully.
1) Band size - theoretical vs actual:
Gamma Sarcoglycan can be highly glycosylated and this types of post-translational modification can contribute to band size shift on the blot. This is very likely the reason for the higher band size than the expected one in C2C12 cells what the customer is experiencing.
Could you consult the following website for further information:
http://www.uniprot.org/uniprot/Q13326
The fact that there is indeed signal with ab104478 with cultured cell lines but not with biopsy samples implies that there may be some protein degradation in the human samples. Unfortunately, degradation can occur during operation or even during the transport of the human samples from the operating theatre to the Lab.
2) Lysis buffer and protein degradation:
I would like to clarify that SDS and DTT do NOT prevent protein degradation; they are not proteinase inhibitors. SDS is an anionic detergent applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. DTT which is dithiothreitol (not DDT) is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size (like ß-mercaptoethanol ).
Please take a look at the Protocol section for further details on the different proteinase inhibitors and the function of SDS and DTT:
https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1
https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A6
It would be nice to see the WB image for Alpha and Delta Sarcoglycan and also for loading control. Could you please attach it to you response?
3) Concentration of protein:
It would be advisable to quantify the protein and load at least 20-30 ug per lane. If it is not possible than I would suggest loading 2x or 4x more than originally to see often intensity of the signal will change or not.
4) Initially, the complaint has been assigned to one product only ab104478. It is not clear which product is causing the problem. Is it only ab104478 or both ab104478 and ab55683?
- Abcam catalogue no: ab104478

- Abcam order number (not required): 1016508 20.01.2012
5) In case both antibodies failed to work, could you please confirm the batch number of ab55683 and indicate if they were shipped together in the same package or not?
If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

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.

I would like to reassure you that ab104478 is tested and covered by our 6 month guarantee for use inWB and human (not mouse) samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab104478.

1.) I suggest to measure the protein content that is loaded per pocket. gamma Sarcoglycan seems to be only weakly expressed and we used 50ug to achieve a positive result in human kidney lysate.

2.) How was the skeletal muscle lysate prepared? The questionnaire indicates that is was boiled in the sample buffer? I suggestto prepare a lysate first by homogenization in RIPA buffer and than dilute the appropriate amount of protein with sample buffer.

3.) Did the loading control work?

4.) Is the secondary antibody working?

5.) When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : https://www.abcam.com/ab9385 .

6.) Could you confirm that a reducing agent (DTT) was used in the sample buffer but non was used when running the gel and also a non-reducing gel was used?

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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Question

Hi,



Here is the complain of two antibodies with same protocol. So I have sent for only for one item .



1) Abcam catalogue no:







Ab104478











2) a) Abcam order number (not required):







1016508

20.01.2012











b) Abcam product lot number:











GR37120-3











3) Description of the problem (high background, wrong band size, more bands, no band etc):







No bands











4) Antibody storage conditions (temperature/reconstitution etc):







-200C







5) a)Sample (Species):







Human skeletal muscle








b) Type of sample (Cell extract/Nuclear extract/Purified protein/Recombinant protein etc).:







Human skeletal muscle











6) a) Sample preparation:







Lysis buffer



Tris HCl,Glycine,Glycerol,SDS,Bromophenol blue





Protease inhibitors



No





Phosphatase inhibitors



No





Reducing agent



Yes, DTT





Boiling for ≥5 min?



5 min











b) Amount of protein loaded:







Protein loaded ug/lane or cells/lane



20µl



















7) a) Electrophoresis/Gel conditions:







Reducing or Non Reducing gel



Non reducing





Percentage of gel



12%





Volts applied



150v





Time applied



1hr 20 min































b) Transfer or blocking conditions:







Type of membrane



PVDF





Protein transfer verified



Yes





Blocking agent and concentration



Milk powder,5%





Blocking time



1 hr





Blocking temperature



Room temperature















































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











Concentration or dilution



1-13µg/ml





Diluent buffer



PBST





Incubation time



Overnight





Incubation temperature



40C





Washing: Buffer Used



PBST





Number of washes



5











9) Secondary antibody:











Species



Goat





Reacts against



Rabbit





Concentration or dilution



1:1000





Diluent buffer



PBST





Incubation time



2 hrs





Incubation temperature:



Room Temperature





Fluorochrome or enzyme conjugate



Enzyme conjugate





Washing: Buffer Used



PBST





Number of washes



5















10) Detection method (ECL, ECL plus, etc.):











ECL











11) Positive and negative controls used:











Positive control



Yes





Negative control



NO









12) a) How many times you have run this staining?







06







b) Do you obtain the same results every time?







Yes







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







Samples and antibody dilution





Regards

Read More
Answer

Thank you for your enquiry regarding ab104478 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.
After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:
1) It looks that the antibody has worked in mouse C2C12 cell line, there is a band on the blot. Though the MW markers are missing so it is difficult to know for certain if the band appears at the right size or not.
2) Are the human samples from biopsies or from postmortem specimen? Could you please confirm? Since the lysis buffer did not contain any protease inhibitors, it may well be that the target is degraded in the human samples. Could you please advise your customer to use a lysis buffer (i.e. RIPA) which contains a mixture of different proteinase inhibitors to prevent the degradation of the protein.
3) Protein concentration:
I would recommend quantifying the total concentration of the protein in the samples and load 20-25 ug per lane. Each sample may have different protein concentration (depending on the mass of starting mater and the volume of the lysis buffer). The volume size 20µl does not guarantee that enough protein is loaded onto the gel and the separated proteins are at detectable levels.
4) Positive control:
Could you specify the positive control run on the same gel? As the on-line datasheet suggests Human kidney tissue lysate or transfected 293T cell line can be applied for this control. Has the customer utilised any of these?
I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

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