Overview

  • Product name
    Anti-E Cadherin antibody [HECD-1]
    See all E Cadherin primary antibodies
  • Description
    Mouse monoclonal [HECD-1] to E Cadherin
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, ICC, IP, IHC-P, IHC-Fr, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Rat, Human
    Does not react with: Mouse
  • Immunogen

    Breast tumor cell line MCF-7

  • Positive control
    • WB: MDA-MB-468 and LNCaP whole cell lysates; MCF7 cell lysate. ICC/IF: Human renal tubular epithelial cells; Human nasal epithelial cells; NSCLC cells; Human renal proximal tubular cells; LNCaP cells. IHC-P: Breast cancer tissue; Human stomach tissue. Flow Cyt: MCF7 and A431 cells. IHC-Fr: Human skin tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab1416 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
Flow Cyt 1/100.

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

ICC Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
IHC-P 1/50 - 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-Fr 1/50 - 1/100.
ICC/IF Use at an assay dependent concentration. PubMed: 19244118
WB 1/50.

Target

  • Function
    Cadherins are calcium-dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types. CDH1 is involved in mechanisms regulating cell-cell adhesions, mobility and proliferation of epithelial cells. Has a potent invasive suppressor role. It is a ligand for integrin alpha-E/beta-7.
    E-Cad/CTF2 promotes non-amyloidogenic degradation of Abeta precursors. Has a strong inhibitory effect on APP C99 and C83 production.
  • Tissue specificity
    Non-neural epithelial tissues.
  • Involvement in disease
    Defects in CDH1 are the cause of hereditary diffuse gastric cancer (HDGC) [MIM:137215]. An autosomal dominant cancer predisposition syndrome with increased susceptibility to diffuse gastric cancer. Diffuse gastric cancer is a malignant disease characterized by poorly differentiated infiltrating lesions resulting in thickening of the stomach. Malignant tumors start in the stomach, can spread to the esophagus or the small intestine, and can extend through the stomach wall to nearby lymph nodes and organs. It also can metastasize to other parts of the body. Note=Heterozygous germline mutations CDH1 are responsible for familial cases of diffuse gastric cancer. Somatic mutations in the has also been found in patients with sporadic diffuse gastric cancer and lobular breast cancer.
    Defects in CDH1 are a cause of susceptibility to endometrial cancer (ENDMC) [MIM:608089].
    Defects in CDH1 are a cause of susceptibility to ovarian cancer (OC) [MIM:167000]. Ovarian cancer common malignancy originating from ovarian tissue. Although many histologic types of ovarian neoplasms have been described, epithelial ovarian carcinoma is the most common form. Ovarian cancers are often asymptomatic and the recognized signs and symptoms, even of late-stage disease, are vague. Consequently, most patients are diagnosed with advanced disease.
  • Sequence similarities
    Contains 5 cadherin domains.
  • Post-translational
    modifications
    During apoptosis or with calcium influx, cleaved by a membrane-bound metalloproteinase (ADAM10), PS1/gamma-secretase and caspase-3 to produce fragments of about 38 kDa (E-CAD/CTF1), 33 kDa (E-CAD/CTF2) and 29 kDa (E-CAD/CTF3), respectively. Processing by the metalloproteinase, induced by calcium influx, causes disruption of cell-cell adhesion and the subsequent release of beta-catenin into the cytoplasm. The residual membrane-tethered cleavage product is rapidly degraded via an intracellular proteolytic pathway. Cleavage by caspase-3 releases the cytoplasmic tail resulting in disintegration of the actin microfilament system. The gamma-secretase-mediated cleavage promotes disassembly of adherens junctions.
  • Cellular localization
    Cell junction. Cell membrane. Endosome. Golgi apparatus > trans-Golgi network. Colocalizes with DLGAP5 at sites of cell-cell contact in intestinal epithelial cells. Anchored to actin microfilaments through association with alpha-, beta- and gamma-catenin. Sequential proteolysis induced by apoptosis or calcium influx, results in translocation from sites of cell-cell contact to the cytoplasm. Colocalizes with RAB11A endosomes during its transport from the Golgi apparatus to the plasma membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • Arc 1 antibody
    • CADH1_HUMAN antibody
    • Cadherin 1 antibody
    • cadherin 1 type 1 E-cadherin antibody
    • Cadherin1 antibody
    • CAM 120/80 antibody
    • CD 324 antibody
    • CD324 antibody
    • CD324 antigen antibody
    • cdh1 antibody
    • CDHE antibody
    • E-Cad/CTF3 antibody
    • E-cadherin antibody
    • ECAD antibody
    • Epithelial cadherin antibody
    • epithelial calcium dependant adhesion protein antibody
    • LCAM antibody
    • Liver cell adhesion molecule antibody
    • UVO antibody
    • Uvomorulin antibody
    see all

Images

  • Anti-E Cadherin antibody [HECD-1] (ab1416) at 1/50 dilution + LNCaP (human prostate cancer cell line) whole cell lyate

    See Abreview

  • All lanes : Anti-E Cadherin antibody [HECD-1] (ab1416) at 1/1000 dilution

    All lanes : MDA-MB-468 cells whole cell lysate

    Lysates/proteins at 80 µg/ml per lane.

    Secondary
    All lanes : HRP-conjugated polyclonal donkey anti-mouse IgG

    Performed under reducing conditions.

    Observed band size: 110 kDa
    why is the actual band size different from the predicted?


    Exposure time: 30 seconds

    See Abreview

  • ab1416 staining E cadherin in Human renal tubular epithelial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 1% BSA for 1 hour at 20°C. Samples were incubated with primary antibody (1/200 in PBS + 0.01% BSA) for 24 hours at 4°C. An Alexa Fluor® 555-conjugated Donkey anti-mouse IgG polyclonal was used as the secondary antibody (1/500).

    See Abreview

  • Breast cancer stained with E Cadherin mouse antibody ab1416

  • Lanes 1-2 : Anti-E Cadherin antibody [HECD-1] (ab1416) at 1/50 dilution
    Lanes 3-4 : Anti-GAPDH at 1 µg/ml

    Lanes 1 & 3 : MDA-MB-231 cell lysate
    Lanes 2 & 4 : MCF-7 cell lysate


    Blocking buffer: 3% milk

  • Overlay histogram showing MCF7 cells stained with ab1416 (red line). The cells were fixed with 80% methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab1416, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H+L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.
  • ab1416 staining E Cadherin in Human nasal epithelial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed paraformaldehyde and permeabilized with Triton X-100 0.1%. Samples were incubated with primary antibody (1/50) for 18 hours at 4°C. A Cy2®-conjugated Horse anti-mouse monoclonal(1/100) was used as the secondary antibody.

    See Abreview

  • Flow cytometric analysis of A431 (human epidermoid carcinoma cell line) cell line labeling E Cadherin with ab1416 at 1/100 dilution (2) compared with an isotype control (1).

    See Abreview

  • ab1416 staining E Cadherin in human normal skin tissue sections by Immunohistochemistry (frozen sections). Tissue was fixed with acetone. Samples were blocked with 10% serum for 30 minutes at 21°C followed by incubation with the primary antibody at a 1/50 dilution for 30 minutes at 21°C. An undiluted HRP-conjugated goat anti-mouse IgG polyclonal was used as secondary antibody.

    See Abreview

  • ab1416 staining E Cadherin in the Human NSCLC cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton in PBS and blocked with 5% serum for 1 hour at room temperature. Samples were incubated with primary antibody (1/100 in 0.3% Triton, 5% Goat Serum in PBS) for 1 hour. An Alexa Fluor® 555-conjugated Goat polyclonal was used as the secondary antibody (1/500).

    See Abreview

  • ab1416 staining E Cadherin in human stomach tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent formaldehyde fixation before heat mediated antigen retrieval in Citrate pH 6.0 and then blocking with 5% serum for 1 hour at 23°C was performed. The primary antibody was diluted 1/100 and incubated with sample for 1 hour at 23°C. A HRP conjugated goat polyclonal to mouse IgG was used undiluted as secondary antibody.

    See Abreview

  • ab1416 staining E Cadherin in Human Renal Proximal Tubular cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton ×100 and blocking with 1% BSA was done for 30 minutes at 210C. Samples were incubated with primary antibody (1/50: in 0.1 % BSA in PBS) for 24 hours at 4°C. An Alexa Fluor®488-conjugated goat polyclonal to mouse IgG was used as secondary antibody at 1/1000 dilution.

    See Abreview

  • Formaldehyde-fixed, 0.2% Triton X-100 permeabilized LNCaP (human prostate cancer cell line) cells stained for E Cadherin (green) using ab1416 at 1/50 dilution in ICC/IF. Blue: Hoechst 33342 staining.

    See Abreview

  • Immunohistochemistry of breast carcinoma staining E Cadherin with ab1416 at 5μg/ml

References

This product has been referenced in:
  • Rhys AD  et al. Loss of E-cadherin provides tolerance to centrosome amplification in epithelial cancer cells. J Cell Biol 217:195-209 (2018). WB, IF ; Human . Read more (PubMed: 29133484) »
  • Kaplan N  et al. EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling. Invest Ophthalmol Vis Sci 59:393-406 (2018). Read more (PubMed: 29351356) »
See all 167 Publications for this product

Customer reviews and Q&As

31-40 of 60 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (prostate cancer cells)
Loading amount
30 µg
Specification
prostate cancer cells
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 27 2012

Question

Hello,
Thank you for your response. I am including my answers to your questionnaire
below.
I am most interested in obtaining the specific banding pattern from a monoclonal
antibody specific to the N-terminus of E-cadherin. In order to ascertain if my
band at 55kDa is specific or not, I would like to re-run my experiment with a
blocking peptide. Do you sell an antibody which meets my specifications and is
also available with a blocking peptide.
Thank you again for your assistance,

1) Abcam product code ab1416
2) Abcam order reference number or product batch number
3) Description of the problem
Although bands are only described in the data sheet at 120kDa, this is obtained
from cell lysate. When serum is used under reducing conditions, multiple bands
are seen both with HECD-1 clone monoclonal antibody, as well as with a
polyclonal antibody. In particular, 120kDa, 97kDa, 80kDa and 55kDa
predominate. Although 97 and 80 have been frequently reported in the
literature as proteolytic fragments, 55 has not. I want to determine if this
band is a specific protein fragment or artifact.
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): Diluted
human serum, also have used albumin depleted human serum
Lysis buffer
Protease inhibitors:
Phosphatase inhibitors
Reducing agent Betamercaptoethanol
Boiling for ≥5 min? yes/no yes
Protein loaded ug/lane or cells/lane 40ug/lane
Positive control Placental lysate
Negative control No primary antibody
5) Percentage of gel 10%
Type of membrane Nitrocellulose and PVDF both used with similar result
Protein transfer verified Yes
Blocking agent and concentration TBS/Tween 5% milk
Blocking time 1 hr
Blocking temperature Room temperature
6) Primary antibody (If more than one was used, describe in “additional notes”)
: ab1416, and a polyclonal was also used
Concentration or dilution 1: 1000
Diluent buffer TBS/Tween with 5% milk
Incubation time: overnight
Incubation temperature: 4 degrees
7) Secondary antibody:
Species: goat
Reacts against: mouse
Concentration or dilution 1:5000
Diluent buffer TBS/Tween with 5% milk
Incubation time 1 hr
Incubation temperature: room temperature
Fluorochrome or enzyme conjugate: attempted both HRP conjugated secondary
antibody, and also biotynylated secondary antibody followed by incubated for 1
hr with 1:8000 Streptavidin-HRP at room temperature
8) Washing after primary and secondary antibodies:
Buffer TBS/Tween 20
Number of washes 5x5 min
9)Detection method
10) How many times have you run this staining? 4-5
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody? As
noted above
Document attachment: Attaching images of your blot is strongly recommended and
can greatly speed up our investigation of your problem.
Not easily technically possible at this time.

Read More
Answer

Thank you for sending the filled-in questionnaire.
After reviewing your protocol, I agree with you that the band at 55kDa is most probably a biological (and therefore interesting) phenomenon.
We do offer a polyclonal antibody against the extracellular domain of E-cadherin that also has a blocking peptide readily available:
ab77287 (Anti-E Cadherin antibody) https://www.abcam.com/index.html?datasheet=77287 https://www.abcam.com/index.html?datasheet=77287.
ab87418 (E Cadherin peptide) https://www.abcam.com/index.html?datasheet=87418 https://www.abcam.com/index.html?datasheet=87418.
Please follow the above link to review the datasheets. Interestingly the blot on the datasheet of ab77287 also indicates a band at ca. 55kDa.
Please let me know if you have any more questions. I hope the above antibody and peptide will help explain the results of your experiments.

Read More

Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining a clear results from this antibody.
Unfortunately, we cannot offer a blocking peptide since the immunogen was not a recombinant peptide but the breast tumor cell line MCF-7.
I am currently trying to find out if the epitope the antibody binds was narrowed down in any way and will let you know as soon as possible.
We will happily offer technical support and in the event that a product is not functioning in the applications 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.
If you would like our help to trouble shoot this problem, I have attached a questionnaire. I would appreciate if you could complete this. It will help you put the information we require together very easily.
I would appreciate if you could also provide an image which would help us to assess the results.
Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.

Read More

Answer

Thank you for contacting Abcam. For ab6328, I would recommend using ab970, the Trypsin Enzymatic Antigen Retrieval Solution that you mentioned. I believe that this will give you very good results in your IHC staining. For ab1416, from what I have read, it look as though that heat mediated antigen retrieval is recommended. You could use ab972, the Heat Mediated High pH Antigen Retrieving Solution, the protocol for this product is available on the website. If there is anything else I can help you with, please let me know.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Skin)
Specification
Skin
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 4% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 08 2011

Question

DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE •Species: human cell line •What’s cell line or tissue: human HCC cell line ex:huh7 hucct1 •Cell extract or Nuclear extract: cell extract PRIMARY ANTIBODY •Reacts against:anti-E cadherin •At what dilution(s) have you tested this antibody:1:1000 •What dilution buffer was used: 5% skim milk in PBST •Incubation time: overnight >16hr •Incubation temperature: 4℃ •What washing steps were done:4 times, 10min every time DETECTION METHOD ECL ANTIBODY STORAGE CONDITIONS -20℃ SAMPLE PREPARATION •what lysis buffer was used: TPER+phospho stop+EDTA free •What protease inhibitors were used: phospho stop+EDTA free •What loading buffer was used: •Phosphatase inhibitors •Did you heat the samples: temperature and time: 95℃ 5min AMOUNT OF PROTEIN LOADED 66μg ELECTROPHORESIS/GEL CONDITIONS •Gel percentage : 6 TRANSFER AND BLOCKING CONDITIONS •Transfer conditions:PVDF, 100volt 90min •Buffer:5% skim milk in PBST •Blocking agent: milk, BSA, serum, what percentage: 5% skim milk •Incubation time:1hr •Incubation temperature:room temperature SECONDARY ANTIBODY •Reacts against:anti-mouse IgG •At what dilution(s) have you tested this antibody: 1:10000 •Incubation time: 1hr •Wash steps: 4 times, 10min every time •Fluorochrome or enzyme conjugate:HRP HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Protein loaded (from20μg to 66μg), but results are still have no band

Read More
Answer

Thank you for your enquiry regarding ab1416 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. The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve your results and that you could consider trying: - Human breast cancer cell lysates as positive control - 1/1000 dilution should work however trying 1/500 dilution could also help. Let me know if the results do not improve, I will then send you a free of charge replacement of this antibody from different lot.

Read More

Question

Here you are my answers to abcam in details regarding my project so please forward to them and hopfuly I can get their guide asap. I am isolating human hertwig's epithelial root sheath (HERS) cells from periodontal ligament (PDL). So the cells that I am isolating are epithelial that should express these two epithelial markers as published in the literature (E-cadherin and Pan-cytokeratin) and will compare them with Stem cells from Human exfoliated deciduous teeth (SHED) using another two anibodies (CD44) that should be highly expressed in SHED than HERS and (CD73) that will be expressed similarly in both cells. So for characterization I ordered these antibodies from abcam: 1-      Mouse monoclonal (HECD-1) to E Cadherin (ab1416) 2-      Mouse monoclonal (PCK-26) to pan Cytokeratin (ab6401) 3-      Mouse monoclonal (F10-44-2) to CD44 (ab6124) 4-      Mouse monoclonal (7G2) to CD73 (ab54217) -          Immunofluorescence staining steps (first time) HERS were cultured in 4 well culture slide and the second day we did the following: a-      Discard the medium b-     Wash 3 times with PBS each time two mins c-      Add 500 µL methanol to each well for 20 mins at 4°C d-     Discard methanol and wash 3 times with PBS each time two mins e-      Add 7 drops of blocking reagent (from Millipore/ comes in the kit of immunoperoxidase secondary detection system) keep it at room temperature for 1 hr. f-       Discard blocking reagent and add the primary antibodies (1:50 ratio) (each antibody was applied separately in each well of the same chamber slide) (we diluted the antibodies using PBS) g-      Keep the chamber slide in 4°C for overnight. h-     Then discard the antibodies, wash 3 times with PBS each time two mins i-        Add 500 µL of secondary antibody (anti mouse FITC) to each well and keep it for 1 hr at room temperature j-       Discard secondary antibody and wash 3 times with PBS each time two mins k-     Add 500 µL of DAPI to each well keep it for 1 hr in a dark room l-        Discard DAPI and wash properly with PBS m-   The cover of the chamber slide was chopped and fluorescence mounting medium was applied (from Dako: S302380) n-     Cover slip was placed and the observed under fluorescence microscope image analyzer Under microscope: We observed the following (as I sent you previously some images); Green cells with green background and blues nucleus. So how to get black background with only green cells and blue nucleus as we got used for immunofluoescence images? -          We did optimize the method (second time) using the same previous procedures but with primary antibodies 1:300 but we got the same result. -          We did optimize the method (third time) using the same previous steps but with these changes: a-      We used PBS with Tween-20 for proper washing b-     Apply methanol and keep it in -20°C for 5 mins c-      Primary antibodies were applied with ratio (1:500) and keep it for 9 hrs at 4°C d-     DAPI was applied for half hr in a dark room. -          But under microscope we got the same result. So we are planning to use primary antibodies 1:1000 and keep it for 3 hrs but we do really need your recommendation and help to get the correct result before we run the test. And waiting for your new quotation please as I did email you. Best regards,    

Read More
Answer

Thank you for contacting us. The background should be black. I am sorry we never had a question like this before. It looks like this problem might be due to instrumentation then antibodies itself. I have following recommendations that might help; - Use less number of cells per well. - Check the viability and passage level of cells. - The cells must be healthy with low number of passages. - Avoid drying out plate between washings. - Check microscope settings. - Check if the blocking is sufficiently done. You can also use 3-5% BSA for 1-2 hour in PBST for blocking. - Use more diluted primary antibodies. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

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Answer

Thank you for contacting us in regards to Anti-E Cadherin antibody (ab1416). Unfortunately we do not have any information on how this antibody affects the E cadherin itself or if it interferes with the internalisation. Experiments to determine this has not been performed by us. I'm sorry that I could not be of more help. If you have any further questions please do not hesitate to ask.

Read More

Answer

Thank you for taking time to complete the questionnaire and reporting this problem to us. 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 some further details: 1. what species of sample is being used? 2. after fixation is a washing step being performed? I would also like to provide some suggestions which may reduce the non-specificity observed. 1. I would reduce the fixation step to 10 minutes to reduce the chances of overfixation 2. You do not mention how the antibody is diluted but I would include a blocking reagent in the buffer such as 1% BSA 3. as this antibody recognises the extracellular domain of E cadherin I would try remove the permiabilisation step. Otherwise, other customer have had success when using 0.025-0.1% Triton X. 4. blocking may also be more efficient using 10% serum from the host species of the secondary antibody. You may also want to increase the blocking step to 1-2 hours. 5. I would also try the staining with the primary antibody being incubated at 1 hour at room temperature. Should the suggestions not improve the results, please do let me know. 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 hope this information is helpful, and I thank you for your cooperation.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (huh-7 cells)
Total protein in input
1e+006 cells
Specification
huh-7 cells
Immuno-precipitation step
Protein A

Abcam user community

Verified customer

Submitted Aug 18 2011

31-40 of 60 Abreviews or Q&A

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