Overview

  • Product name
  • Description
    Rabbit polyclonal to CTHRC1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ELISA, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Peptide corresponding to the C-terminal of CTHRC1 protein (Human).

  • Positive control
    • KZ-28 cells MMRU cells PMWK cells

Properties

Applications

Our Abpromise guarantee covers the use of ab54181 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 1/1000. Detects a band of approximately 27 kDa (predicted molecular weight: 27 kDa).
ELISA 1/2000 - 1/5000.
IHC-P Use at an assay dependent concentration.

Target

  • Function
    May act as a negative regulator of collagen matrix deposition.
  • Tissue specificity
    Isoform 1 is expressed in calcified atherosclerotic plaque and chondrocyte-like cells.
  • Sequence similarities
    Contains 1 collagen-like domain.
  • Post-translational
    modifications
    N-glycosylated.
  • Cellular localization
    Secreted > extracellular space > extracellular matrix.
  • Information by UniProt
  • Database links
  • Alternative names
    • Collagen triple helix repeat containing 1 antibody
    • Collagen triple helix repeat containing protein 1 antibody
    • Collagen triple helix repeat-containing protein 1 antibody
    • CTHR1_HUMAN antibody
    • Cthrc1 antibody
    • NMTC1 protein antibody
    • Protein NMTC1 antibody
    see all

Images

  • Immunohistochemistry analysis of melanoma tissues using ab54181. Melanoma in situ (MIS) are negative, primary melanoma (PM) are positive and metastatic melanoma (MM) are strongly positive.

References

This product has been referenced in:
  • Eriksson J  et al. Gene expression analyses of primary melanomas reveal CTHRC1 as an important player in melanoma progression. Oncotarget 7:15065-92 (2016). WB, IHC . Read more (PubMed: 26918341) »
  • Kharaishvili G  et al. Collagen triple helix repeat containing 1 protein, periostin and versican in primary and metastatic breast cancer: an immunohistochemical study. J Clin Pathol : (2011). IHC-P ; Human . Read more (PubMed: 21742751) »
See all 2 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Thank you for your reply.

I have recieved the general WB testing protocol used by our source for this antibody. Please note this is a guideline only, and may require some individual optimization.
I hope this will be helpful and look forward to hearing from you with the results of the next experiment. Please do not hesitate to contact me withfurther detailsif the results do not improve.

Western Blot S.O.P.
Always plan when you are going to do Western Blot, and prepare the gels beforehand

Setting Up the Bio-Rad Gel Moulding Apparatus:

1. Ensure stacking plates are free of chips and/or cracks, especially along the bottoms.
NOTE: there are various sizes of stacking plates so make sure you have the correct one (ie 1.5mm)
2. Clean the glass plates thoroughly first with Windex1 then with 70% ethanol1
3. On a flat surface, place plates together, slide into green holder and clamp shut.
4. Examine bottom and see if plates are aligned properly. If the plates do not align, the gel will leak.

Making and Setting the Separating Gel:

1. Follow the chart to make Separating Gel (makes 20mL enough for two 1.5mm gels):

Acryl Percentage 12%
30% Acryl stock 8 ml
4x Separate buffer 5 ml
ddH2O 7 ml
TEMED 10 ul
10%APS 75 ul

Acryl Percentage 10%
30% Acryl stock6.7 ml
4x Separate buffer 5 ml
ddH2O8.3 ml
TEMED 10 ul
10%APS 75 ul

Acryl Percentage 8%
30% Acryl stock 5.4 ml
4x Separate buffer 5 ml
ddH2O 8.3 ml
TEMED 10 ul
10%APS 75 ul

Acryl Percentage 6%
30% Acryl stock4 ml
4x Separate buffer 5 ml
ddH2O 9.6 ml
TEMED 10 ul
10%APS 75 ul

NOTE: The percentage of acrylamide for separating gel depends on the size of your targetprotein. Generally the bigger the protein, the smaller percentage of acrylamide is used, and vice versa. The most commonly used is 10%.

NOTE: 10% APS once made should be consumed within 1 month (storing at 4oC). New APS can be made using 1 tablet of APS plus 1mL of ddH2O.
Ammonium Persulfate tablets (100mg),
* CAUTION acrylamide is a potent neurotoxin so use with extreme care*

2. Add ddH2O, 4x Separation Buffer and 30% Acrylamide Stock into a 50mL Falcon
tube. The same glass pipette can be used for ddH2O then 4x Separation Buffer

3. Add TEMED and 10% APS, and mix thoroughly without creating bubbles.

4. Use a 5mL glass pipette to take up about 7.0mL of gel mixture and add to each
mould, adding into the corner to prevent bubble formation up to just above the upper
bar of the green clamp

5. Overlay with 1mL of butanol using disposable plastic pipette and allow about 45
minutes to an hour to set before making the Stacking Gel.

Making and Setting the Stacking Gel (30 minutes):

1. Remove the butanol sitting on top of the separating gel by drawing the liquid up with a piece filter paper without contacting the gel

2. Follow the chart to make 4% Stacking Gel (makes 5mL enough for two 1.5mm gels):
30% Acryl Stock 670uL
4x Stack Buffer 1.25mL
ddH2O 3.05mL
TEMED 10uL
10% APS 20uL

3. Add ddH2O, 4x Stacking Buffer and 30% Acrylamide Stock into a 10mL Falcon tube.
The same glass pipette can be used for ddH2O then 4x Stocking Buffer

4. Add TEMED and 10% APS, and mix thoroughly without creating bubbles.

5. Use a disposable plastic pipette to add gel up to the top of each gel, again adding to corner.

6. Carefully insert comb in mould, do not get your face too close as it has a tendency to splash out excess gel mixture and try to avoid creating air bubbles

7. Let set about 35 minutes.

Storing Gels:

1. Cut a piece of seran wrap

2. Lay two pieces of paper towels on top

3. Use squeeze bottle of H2O to damp the paper towel

4. Place the gel on the wet paper towel and wrap around with the seran wrap

5. Label and store at 4°C up to 1 month

Running the Gels (1.5-2.5 hours):

1. Before loading the samples in the well, always boil the samples for 5 minutes

2. Remove set gels from the clamps, assemble them into the holder and set in tank.

3. Pour 1x Running Buffer into the slot between the gels, filling until it overflows (and
to the designated line. ie 2 gels) then remove the combs.

4. Load approximately 5uL of ladder into an edge well.

5. Load the samples, each well on a 1.5mm gel holds about 30-40uL, be sure not to
overfill, and record each lane’s contents in the lab notebook

NOTE: Try not to load at the edge lanes of the gel, also avoid loading in lanes that are poorly formed (ie uneven bottom)

6. Load 1X protein loading dye1 into all the empty wells to prevent samples from
diffusing into other wells

7. Set lid on tank ensuring correct positive/negative orientation and set to run at 200V
for about 10-15 minutes or until the loading dye hits the stacking/separating gel interphase, check often.

8. Once samples hit the separation gel turn voltage down to 100V and run an additional hour or two depending on size of protein of interest.

NOTE: Be sure to record the exact time used to run the gel in the lab notebook
Transferring the Gels (1-1.5 hours):

1. Ten minutes prior to gel finishing its run, soak the transfer PVDF membrane in
100% Methanol.

NOTE: slide in the PVDF membrane from an angle to avoid any air bubbles and to achieve even distribution of the buffer on the membrane surface

The methanol can then be recycled to make more 1X transferring buffer

10X transferring buffer 100mL
ddH2O 700mL
recycled methanol 200mL

2. Soak the sponge and filter paper in 1X transferring buffer1. Use the 15mL falcon tube to roll out any air bubbles between the filter paper and the sponge

3. Disassemble the gel apparatus; use the plastic wedge to separate the 2 glass pieces.
Be careful not to break the gel

4 Protein Ladder

5. Cut away the stacking gel using the wedge and any excess gel. DO NOT cut away
your protein lanes

6. Soak the gel in the 1X transferring buffer along with the sponge for 10 minutes

7. Assemble the transfer apparatus


NOTE: it is a good idea to always reverse the gel orientation for transfer such that the orientation on the PVDF membrane is always the same as the gel loading orientation

8. Place assembled holder into tank, ensure charges are lined up correctly; otherwise
protein will move out of gel the wrong direction and be lost!

9. Obtain the ice pack from the -20° C fridge3 and place in the tank as well to prevent overheating of the apparatus

10. Allow to run at 100V for 60 minutes on stirring block
Visualization of PVDF membrane using Ponceau S solution
The PVDF membrane can be visualized using Ponceau Staining to observe the transfer of the gel

Ponceau S solution Recipe:
10ml MiliQ water
0.3ml glacial acetic acid
0.033g ponceau S
Final value 30mL top up with water

1. After electrophoresis, immerse the blotted membrane in a sufficient amount of Ponceau S staining solution and stain for 2 minutes. Do this before blocking

During the transfer, prepare blocking solution (3% skim milk in TBS):
Weigh 0.3g of skim milk powder1 for every 10mL of TBS buffer. For 1 gel,
we need a total of ˜30mL of 3% skim milk. If the blocking solution is ready
before the transfer is complete, we can directly put the membrane in blocking
solution and skip step #10.

2. Should be able to see the proteins after 2 minutes, if not, leave it for 5 minutes

3. To wash off the dye: transfer the membrane into water for two washes of 5 minutes each
NOTE: Ponceau S solution can be reused

4. Once the dye has came off, remove the membrane and block as normal

Blocking and Probing (4 hours):

1. After transfer dissemble the apparatus. Check if the transfer is complete (ie ladder
bands should be on the membrane instead of the gel)

2. Cut away any excess membrane to decrease the surface area of the membrane

3. Cut one corner of the membrane to mark the orientation. (ie the upper left corner of
the ladder)

4. Move the membrane to blocking solution and set on shaker (low; or setting 2) for 30 minutes

5. Remove from blocking solution and place in milk solution containing diluted
primary antibody (1:1000 = 10ul Ab for 10mL of milk solution) for 1 hour on shaker
(at setting 2; low) at room temperature, or overnight at 4° C

6. Remove from the Ab solution and wash once briefly and once thoroughly in TBS for
5 minutes on shaker (setting to 4; high). Meanwhile prepare the secondary Ab
solution

7. Remove from blocking and place in milk solution containing diluted secondary
antibody (1:1000) for 30 minutes on shaker (at setting 2; low) at room temperature.

8. Remove from the Ab solution and wash twice briefly and once thoroughly in TBS
once for 5 minutes and proceed to which ever imaging method you are using.

Imaging using X-ray ECL (20-30 minutes):

1. Mix equal amount of ECL solution 5 1 with ECL solution 2 (ie one membrane uses
approximately 2mL of the combined solution)

2. Set the membrane on a seran wrap facing up (ie cut should be at upper left corner of the ladder)

3. Using the disposable plastic pipette, put ECL solution onto the membrane and let it
sit for 1 minute

4. Dab the membrane on a paper towel to remove excess ECL solution, and place the
membrane onto a second seran wrap with the right side facing down

5. Wrap the membrane in seran wrap and place membrane into the metal frame under
the plastic covering. Make sure you roll out any air bubbles between the membrane
and the plastic for a clearer image.

6. Prepare the Exposing Solutions:

a. Developer; Make 500mL at 1:4 dilutions (400mL ddH20 to 100mL developer
concentrate).

b. Fixer - Make 500mL at 1:4 dilutions (400mL ddH20 to 100mL fixer concentrate).

c. Fill a third container with tap water as a rinse.

7. Take the solutions, some forceps, scissors, a timer, the metal frame, x-ray film, and a timer into the dark room. (DO NOT EXPOSE THE X-RAY FILM TO LIGHT!)

8. CLOSE THE DOOR AND TURN OFF THE LIGHT.
In the Dark:

9. Cut a piece of x-ray film to fit just a little over your blots.

10. Place the film over the membrane, and close the frame tightly using the locking
mechanism. Expose the film to the membrane for 1 minute using preset timer.
NOTE: If the resulting film has a weak signal, you can try again with a longer time

11. While the film is exposing, make sure to return the remaining x-ray film to the box
and close the box.

12. After the exposure, remove the film from the frame, and place it in the developer
solution until bands appear. Make sure to let all the bands come out prior to fixing
the film. (Usually until counting to 10-12 results in most clear image)

13. Once the bands are seen, quickly move the film to the fixer solution for about 20
seconds, or until film darkens.

14. The film can then be rinsed in the container of water

15. Make sure to check once again that the remaining film is put away correctly prior to
turning the light back on.

16. Place the film on top of the membrane to mark the ladders at the appropriate spot

If the film:
Looks too dark: try exposing the film to the developer solution for a shorter
amount of time

Has a lot of unspecific binding: return the membrane to wash in TBS for a
longer period

Has weak signal from Ab binding: try exposing the film to the membrane
for a longer period

Stripping the PVDF membrane:
In case other antibodies has to be probed onto the same sample, we can use stripping buffer to return the blot to its unprobed state

Stripping buffer Recipe
2% SDS (stock 10% SDS) 20 mL
100mM beta-mercaptoethanol (stock 1M) 10 mL
50mM Tris, pH 6.8 (stock 500nM, pH 8.0) 10 mL
Adjust pH to 6.8 (˜6 drops of 5M HCl), then add ddH2O to 100

1. Heat stripping buffer to 42° C in water bath

2. Incubate blot with striping buffer at 42° C for 20 minutes with gentle shake from time to time

3. Wash blot several times in 1X TBS, plus one wash for 5 minutes on shaker

4. Blot is now like “after transfer” blot, and can be blocked with 3% milk

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Answer

Thank you for contacting us.

As indicated on the datasheet (https://www.abcam.com/ab54181), the positive controls are melanomacell lines (KZ-28, MMRU, PMWK).
We have the following product your customer may be interested in : Melanoma (Human) Whole Cell Lysate - tumor tissue (https://www.abcam.com/ab29339).

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.

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

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Question

LOT NUMBER -- NOT SPECIFIED -- ORDER NUMBER 011294-00 DESCRIPTION OF THE PROBLEM 1)Work at the same time with other gene antibody (ISG15) which ISG15 showed a nice significant and specific band but for the CTHRC1 showed: 2)Non-specific band + Multiple band + No signal , no high background Note: both antibody are using the same secondary antibody Goat to Rabbit antibody and same protocol but ISG15 showed good result but CTHRC1 showed bad result SAMPLE oral cancer cell lines total protein extracted from oral cancer cell lines PRIMARY ANTIBODY Manufacturer:abcam species: Rabbit diluent: 5%skim milk Followed the recommended dilution from the data sheet 1/1000, Incubation time:overnight washing step: tbst 10 minute 3 times DETECTION METHOD chemiluminescent HRP substrate POSITIVE AND NEGATIVE CONTROLS USED non ANTIBODY STORAGE CONDITIONS Aliquot to 5uL in pcr tubes and storage at -20 degree celcius SAMPLE PREPARATION protein extraction buffer contain: DTT, Na3VO4, protease inhibitor, and protein lysis buffer 5x loading dye protease inhibitors 1ul heating samples: 15 minute at 95 degree celcius AMOUNT OF PROTEIN LOADED 100ug ELECTROPHORESIS/GEL CONDITIONS 15% reducing gel TRANSFER AND BLOCKING CONDITIONS 5% skim milk (2g skim milk + 40ml tbst) SECONDARY ANTIBODY secondary antibody: followed the recommended dilution from the data sheet: 1/5000 incubation time: HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? blocking period,from 1 hours extented to 2 hours antibody dilution from 1/1000- 1/2000 ADDITIONAL NOTES that is not the secondary antibody and the protocol problems because I work at the same time with two different antibody (ISG15 and CTHRC1) but ISG15 showed a nice significant specific band result but not CTHRC1. both antibody are rabbit polyclonal

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
Having reviewed this case, I would like to confirm a few more details if you wouldn't mind. This will aid in understanding the problems further.
1. I was unable to find the order from the reference that you gave me, did you purchase this antibody through one of our distributors? Do you have an approximate date when the order was placed?
2. Could you let me know what the lot number of your vial was?
3. Would it be possible to share an image of the western blot obtained? Both with CTHRC1 and ISG15? This will help greatly in understanding the problems you have been observing. Do you see any band at the expected 27 kDa?
4. Was the CTHRC1 and ISG15 probed on different membranes or was the same membrane used and stripped? If so how was the membrane treated to perform this process?
5. Was the protein transfer verified by staining of the membrane?
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I look forward to recieving your reply.

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Answer

Thank you for taking the time to contact us. Your enquiry has been forwarded to me as my colleage Anja is currently away from the office. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee forWB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily and I should then be able to provide some further suggestions to you.

I would appreciate if you could also provide an image, includingmolecular weight markers, which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.

Order Details
Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


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


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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