Overview

  • Product name
    Anti-Metallothionein antibody [UC1MT]
    See all Metallothionein primary antibodies
  • Description
    Mouse monoclonal [UC1MT] to Metallothionein
  • Host species
    Mouse
  • Specificity
    Cross reacts with MT1 and MT2.
  • Tested applications
    Suitable for: IHC-Fr, ICC/IF, WB, Flow Cyt, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Horse, Dog, Human, Mummichug fish
  • Immunogen

    Full length protein (cross linked rabbit liver MT1 and MT2).

  • Positive control
    • HeLa cell lysate treated with 100uM CdCl2 Rehydrated rabbit liver MTI/MTII

Properties

Applications

Our Abpromise guarantee covers the use of ab12228 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
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 - 5 µg/ml.
WB 1/1000. Detects a band of approximately 6-20 kDa (predicted molecular weight: 6 kDa). Please note: often Western blots done on cell lysates with this antibody produce many bands; we suspect that metallothionein binds to many other proteins, thus producing these results. As the predicted MW is around 6 kDa, use 12.5-20% gel and be sure the protein is not run off the gel during electrophoresis.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-P Use at an assay dependent concentration.

Target

Images

  • Anti-Metallothionein antibody [UC1MT] (ab12228) + Hela cell lysate

    Secondary
    HRP-conjugated antibody.

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 6 kDa


    Exposure time: 2 minutes
  • ab12228 staining human uterus tissue at 10 ug/ml using IHC-P.

  • ICC/IF image of ab12228 stained HeLa 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 (ab12228, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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.

  • Overlay histogram showing HeLA cells stained with ab12228 (red line). The cells were fixed with 100% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab12228, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was Mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Anti-Metallothionein antibody [UC1MT] (ab12228) at 1/1000 dilution + Rabbit liver lysates

    Predicted band size: 6 kDa

  • ICC/IF image of ab12228 stained HeLa 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 (ab12228, 10µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 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.

  • All lanes : Anti-Metallothionein antibody [UC1MT] (ab12228)

    Lane 1 : Marker
    Lane 2 : MTI
    Lane 3 : MTII
    Lane 4 : Mummichug CdCl2

    Predicted band size: 6 kDa

  • ab12228 staining catfish kidney tissue sections by IHC-Fr.  Sections were acetone fixed and blocked with a commercial blocking agent prior to incubation with the primary antibody, diluted 1/50, for 16 hours at 4°C.  An Alexa Fluor® 488 conjugated goat anti-mouse antibody, diluted 1/1000, was used as the secondary.

    See Abreview

References

This product has been referenced in:
  • Lu J  et al. Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm. Stem Cell Res 28:48-55 (2018). ICC/IF ; Human . Read more (PubMed: 29427839) »
  • Borchard S  et al. The exceptional sensitivity of brain mitochondria to copper. Toxicol In Vitro 51:11-22 (2018). Read more (PubMed: 29715505) »
See all 30 Publications for this product

Customer reviews and Q&As

1-10 of 30 Abreviews or Q&A

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (15% acrylamide)
Sample
Human Cell lysate - whole cell (osteosarcoma)
Specification
osteosarcoma
Blocking step
blocking buffer Sigma as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jan 14 2015

Application
Immunohistochemistry (Frozen sections)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10 · Temperature: 23°C
Sample
Rat Tissue sections (Brain)
Specification
Brain
Permeabilization
Yes - Methanol
Fixative
Methanol

Abcam user community

Verified customer

Submitted Oct 27 2014

Answer

Thank you for contacting us.

The reason it comes as an error message is because there is no specific protocol for this antibody. The link to the general protocol is below:

https://www.abcam.com/index.html?pageconfig=resource&rid=11375


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Answer

We do not have any free samples or small samples of any of our antibodies but both ab12228 and ab3125 will be covered by our guarantee for one year if you plan on using them for flow cytometry with samples that are one of the species listed on the datasheets. (Human, ferret, and monkey are listed on the ab3125 datasheet and human, mouse, rat and several others on the ab12228 datasheet).

Note that metallothionein is cytoplasmic, so you will not be able to sort live cells. Antibody ab3125 anti-p75 NGF Receptor, on the other hand, recognizes an extracellular epitope, so you should be able to sort live cells.

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Answer

Thank you for contacting us. 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 AbTrial 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. Or you can find more information about this program at the following link:

www.abcam.com/abtrial

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 your new contact.

My colleague is out of office today. I have attached the Western blot Questionnaire to this e-mail and would be grateful if you could complete it and send it back to us.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

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Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

I have read the details you have kindly provided and will be very happy to investigate the antibody further.

- Please fill the attached questionnaire and provide images?

Thank you very much for your cooperation. I will look forward to hearing from you soon.

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Answer

Thank you for getting back to me.
I think that sounds like a good idea. I would say that trying a 15% or 20% gel would probably perform better. Otherwise, make sure that the protein is not run off the gel (stop it well before the dye front reached the bottom).
I look forward to hearing how you get on. Until then, I hope you have a nice weekend.

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Answer

Thank you for getting back to me with this information and sorry for the delay in my reply.
The reason I asked about the ladder is because the Metallothionein protein is very small and it is therefore important to make sure that the lower end of the protein ladders are detecting the expected region and that the protein has been transfered as expected.
What I would suggest doing in order to make sure the protein is not just simply moving off the gel is to run a higher % gel. We have recommended using a 4-15% gel but you could just make a 15 or 20 % gel which should make the resolution at the lower scale better as well. Just make sure not to run the protein off the gel. If you would prefer, you could always perform a dot blot before to see if there is any positive signal in your sample at all.
The only other thing I noticed was that you are currently using your secondary antibody at a dilution of 1/10,000 but the manufacturer recommends a starting point of 1/2000. It may be worthwhile to increase the concentration of the secondary to this, just to make sure that the signal is as strong as it could be. I would also increase the amount of protein loaded to 30 ug/well and perform a boiling step for 5 minutes following addition of the loading buffer.
Am I correct in thinking that you used CdCl2 with your cells prior to performing the Western blot? This can help in removing the zinc from the MT, mentioned in the following reference:
http://www.ncbi.nlm.nih.gov/pubmed/1779092
Do you have any evidence to suggest your cells should be expressing significant amounts of metallothionein? Using the Gene Expression Atlas I have not found reference to skMDC showing expression and from the following study it seems that skeletal muscle shows quite low levels when not put under stress (exercise):
http://ep.physoc.org/content/90/4/477.full
Do you have access to any other positive controls, such as liver tissue?
I look forward to receiving your reply.

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Question

1) Abcam product code ab : ab1228

2) Abcam order reference number or product batch number GR83765-2
3) Description of the problem
I try to detect Metallothionein in muscle cells with your product ab12228, I follow exactly the protocol given on your web site.
For the blocking step I use Skim milk 5% and I obtained a high background. On the other hand I loaded 10µg of protein and I didn’t observe any signal ( see below).
It cannot be my secondary antibody because I use the same for other dectection with the same protocol and I have good results
4) Sample preparation:
Type of sample: cell lysates
Lysis buffer: Ripa buffer ( thermo-89900)+ Inhibitor cocktail (thermo-78442)+EDTA
Protease inhibitors: in the inhibitor cocktail
Phosphatase inhibitors: in the inhibitor cocktail
Reducing agent:
Boiling for ≥5 min? NO
Protein loaded ug/lane or cells/lane :10µg/lane
Positive control NO
Negative control NO
5) Percentage of gel :4-12% Bis -Tris
Type of membrane : PVDF 0.2µm
Protein transfer verified: Yes by ponceau red
Blocking agent and concentration: 5% skim milf in 1xPBS/0.05% tween20
Blocking time : 1h
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 ab12228 1/100
Diluent buffer Blocking buffer
Incubation time 1h
Incubation temperature: Room temperature
8) Secondary antibody: Goat anti mouse IgG-HRP
Species: Mouse
Reacts against: mouse
Concentration or dilution 1/10 000
Diluent buffer Blocking buffer
Incubation time 1h
Incubation temperature: Room temperature
Fluorochrome or enzyme conjugate: enzyme conjugate
9) Washing after primary and secondary antibodies:
Buffer PBS/0.05% tween20
Number of washes 2x5min after 1st Ab and 3x5min after the 2sd Ab
10)Detection method
ECL+
11) How many times have you run this staining? 3
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody?
Try to increase protein concentration / use 1st Ab at 1/500/ take PVDF membrane 0.2µm/ add 2mM CaCl2 final in transfer buffer as it done in some publication.
Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

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Answer

Thank you for your reply.
I'd like to clarify a few details if you wouldn't mind in order to understand the problems encountered with the Anti-Metallothionein antibody [UC1MT] (ab12228) more fully.
1. You mentioned that with 10 ug loaded there was no signal, does this mean with the 5% milk blocking where you described a high background you loaded a larger quantity of protein?
2. Would you be able to label the ladder of the blot share with me? And provide an image of the blot where you described seeing high background?
3. You mentioned that the secondary antibody has worked well with other proteins, which antibody was used with it to obtain this result?
4. Has any loading control been performed? I.e. blotting if a protein such as GAPDH or actin?
Once I have received this information I should be able to give you a few suggestions in order to try and improve the results currently observed.
I look forward to receiving your reply.

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1-10 of 30 Abreviews or Q&A

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