Recombinant Anti-Collagen I antibody [EPR7785] - BSA and Azide free KO Tested (ab215969)

Product name

Anti-Collagen I antibody [EPR7785] - BSA and Azide free
See all Collagen I primary antibodies
Description

Rabbit monoclonal [EPR7785] to Collagen I - BSA and Azide free
Host species

Rabbit
Specificity

Compared with ab138492, ab255809 has higher affinity. We recommend ab255809 as an alternative for testing pro-Collagen forms in western blot. ab138492 works in western blot in samples with high level of collagen I, like HFF-1, MRC-5, skin tissue etc.

ab138492 is specific for pro-Collagen and 139kda mature from, while ab255809 is specific for pro-Collagen and 35kda C-terminal pro peptide.
Tested applications

Suitable for: IHC-Fr, WB, IHC-P, ICC/IFmore details
Species reactivity

Reacts with: Human
Predicted to work with: Cow
Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: HFF-1 and MRC-5 whole cell lysates, Human stomach, skin and adrenal gland tissue lysates. IHC-P: Human breast carcinoma, colon, placenta and stomach tissues. IHC-Fr: Frozen Human cervix and uterus tissue sections. ICC/IF: U2OS cells
General notes
ab215969 is the carrier-free version of ab138492.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar^® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar^® is a trademark of Fluidigm Canada Inc.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C. Do Not Freeze.
Dissociation constant (K_D)

K_D = 1.22 x 10 ^-10 M
Learn more about K_D
Storage buffer

pH: 7.20
Constituent: 99% PBS
Carrier free

Yes
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR7785
Isotype

IgG
Research areas

Alternative Versions
Compatible Secondaries
- Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
- Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
Conjugation kits
- PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
- APC Conjugation Kit - Lightning-Link® (ab201807)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
Recombinant Protein
- Recombinant Human Collagen I protein (ab158152)
Related Products
- Human Pro-Collagen I alpha 1 ELISA Kit (ab210966)

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab215969 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.
WB		Use at an assay dependent concentration. Predicted molecular weight: 139 kDa. Sample preparation: frozen, ground tissue samples were added to hot lysis buffer (10 mM Tris-HCl (pH 8.0); 1%SDS; 1.0 mM Na-Orthovanadate), boiled for 10-20 minutes and sonicated (3 seconds at 40kW, 30 intervals) prior to centrifugation. Positive Control: Hu stomach, skin and adrenal gland tissue lysates. Acid or enzyme treatment with pepsin is a better method to isolate collagen. Continuous refrigeration throughout collagen extraction is important to avoid degradation and denaturation. Take care with pH, temperature, and concentration to avoid collagen polymerization.
IHC-P		Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
ICC/IF		Use at an assay dependent concentration.

Notes
IHC-Fr Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 139 kDa. Sample preparation: frozen, ground tissue samples were added to hot lysis buffer (10 mM Tris-HCl (pH 8.0); 1%SDS; 1.0 mM Na-Orthovanadate), boiled for 10-20 minutes and sonicated (3 seconds at 40kW, 30 intervals) prior to centrifugation. Positive Control: Hu stomach, skin and adrenal gland tissue lysates. Acid or enzyme treatment with pepsin is a better method to isolate collagen. Continuous refrigeration throughout collagen extraction is important to avoid degradation and denaturation. Take care with pH, temperature, and concentration to avoid collagen polymerization.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
ICC/IF Use at an assay dependent concentration.

Function

Type I collagen is a member of group I collagen (fibrillar forming collagen).
Tissue specificity

Forms the fibrils of tendon, ligaments and bones. In bones the fibrils are mineralized with calcium hydroxyapatite.
Involvement in disease

Defects in COL1A1 are the cause of Caffey disease (CAFFD) [MIM:114000]; also known as infantile cortical hyperostosis. Caffey disease is characterized by an infantile episode of massive subperiosteal new bone formation that typically involves the diaphyses of the long bones, mandible, and clavicles. The involved bones may also appear inflamed, with painful swelling and systemic fever often accompanying the illness. The bone changes usually begin before 5 months of age and resolve before 2 years of age.
Defects in COL1A1 are a cause of Ehlers-Danlos syndrome type 1 (EDS1) [MIM:130000]; also known as Ehlers-Danlos syndrome gravis. EDS is a connective tissue disorder characterized by hyperextensible skin, atrophic cutaneous scars due to tissue fragility and joint hyperlaxity. EDS1 is the severe form of classic Ehlers-Danlos syndrome.
Defects in COL1A1 are the cause of Ehlers-Danlos syndrome type 7A (EDS7A) [MIM:130060]; also known as autosomal dominant Ehlers-Danlos syndrome type VII. EDS is a connective tissue disorder characterized by hyperextensible skin, atrophic cutaneous scars due to tissue fragility and joint hyperlaxity. EDS7A is marked by bilateral congenital hip dislocation, hyperlaxity of the joints, and recurrent partial dislocations.
Defects in COL1A1 are a cause of osteogenesis imperfecta type 1 (OI1) [MIM:166200]. A dominantly inherited connective tissue disorder characterized by bone fragility and blue sclerae. Osteogenesis imperfecta type 1 is non-deforming with normal height or mild short stature, and no dentinogenesis imperfecta.
Defects in COL1A1 are a cause of osteogenesis imperfecta type 2A (OI2A) [MIM:166210]; also known as osteogenesis imperfecta congenita. A connective tissue disorder characterized by bone fragility, with many perinatal fractures, severe bowing of long bones, undermineralization, and death in the perinatal period due to respiratory insufficiency.
Defects in COL1A1 are a cause of osteogenesis imperfecta type 3 (OI3) [MIM:259420]. A connective tissue disorder characterized by progressively deforming bones, very short stature, a triangular face, severe scoliosis, grayish sclera, and dentinogenesis imperfecta.
Defects in COL1A1 are a cause of osteogenesis imperfecta type 4 (OI4) [MIM:166220]; also known as osteogenesis imperfecta with normal sclerae. A connective tissue disorder characterized by moderately short stature, mild to moderate scoliosis, grayish or white sclera and dentinogenesis imperfecta.
Genetic variations in COL1A1 are a cause of susceptibility to osteoporosis (OSTEOP) [MIM:166710]; also known as involutional or senile osteoporosis or postmenopausal osteoporosis. Osteoporosis is characterized by reduced bone mass, disruption of bone microarchitecture without alteration in the composition of bone. Osteoporotic bones are more at risk of fracture.
Note=A chromosomal aberration involving COL1A1 is found in dermatofibrosarcoma protuberans. Translocation t(17;22)(q22;q13) with PDGF.
Sequence similarities

Belongs to the fibrillar collagen family.
Contains 1 fibrillar collagen NC1 domain.
Contains 1 VWFC domain.
Post-translational
modifications

Proline residues at the third position of the tripeptide repeating unit (G-X-Y) are hydroxylated in some or all of the chains. Proline residues at the second position of the tripeptide repeating unit (G-X-Y) are hydroxylated in some of the chains.
O-linked glycan consists of a Glc-Gal disaccharide bound to the oxygen atom of a post-translationally added hydroxyl group.
Cellular localization

Secreted > extracellular space > extracellular matrix.
Target information above from: UniProt accession P02452 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 282187 Cow
- Entrez Gene: 282188 Cow
- Entrez Gene: 1277 Human
- Entrez Gene: 1278 Human
- Omim: 120150 Human
- Omim: 120160 Human
- SwissProt: P02453 Cow
- SwissProt: P02465 Cow
- SwissProt: P02452 Human
- SwissProt: P08123 Human
- Unigene: 172928 Human
- Unigene: 681002 Human
see all
Alternative names
- Alpha 1 type I collagen antibody
- Alpha 2 type I collagen antibody
- alpha 2 type I procollagen antibody
- alpha 2(I) procollagen antibody
- alpha 2(I)-collagen antibody
- Alpha-1 type I collagen antibody
- alpha1(I) procollagen antibody
- CO1A1_HUMAN antibody
- COL1A1 antibody
- COL1A2 antibody
- collagen 1 antibody
- collagen alpha 1 chain type I antibody
- Collagen alpha-1(I) chain antibody
- collagen alpha-1(I) chain preproprotein antibody
- Collagen I alpha 1 polypeptide antibody
- Collagen I alpha 2 polypeptide antibody
- collagen of skin, tendon and bone, alpha-1 chain antibody
- collagen of skin, tendon and bone, alpha-2 chain antibody
- Collagen type I alpha 1 antibody
- Collagen type I alpha 2 antibody
- EDSC antibody
- OI1 antibody
- OI2 antibody
- OI3 antibody
- OI4 antibody
- pro-alpha-1 collagen type 1 antibody
- type I proalpha 1 antibody
- type I procollagen alpha 1 chain antibody
- Type I procollagen antibody
see all

Western blot - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

All lanes : ab138492 and ab255809 at 1/1000 dilution

Lane 1 : A549 (Human lung carcinoma epithelial cell) whole cell lysate
Lane 2 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) supernatant lysate
Lane 4 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate
Lane 5 : SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 6 : Human lung tissue lysate
Lane 7 : Human liver tissue lysate
Lane 8 : HFF-1 (Human Skin fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 139 kDa
Observed band size: 220 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM/TBST.

Exposure Time: Lane 1-7: 180 seconds, Lane 8: 60 seconds.

ab181602 was used as loading control.

Compared with ab138492, ab255809 has higher affinity. We recommend ab255809 as an alternative for testing pro-Collagen forms in western blot.

ab138492 is specific for pro-Collagen and 139kda mature from, while ab255809 is specific for pro-Collagen and 35kda C-terminal pro peptide.

For better using ab255809, we recommend loading higher amount of lysate or using lower antibody dilution.

This data was developed using ab138492, the same antibody clone in a different buffer formulation.
Immunocytochemistry/ Immunofluorescence - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

This data was developed using the same antibody clone in a different buffer formulation (ab138492). ab138492 staining Collagen alpha-1 chain in wild-type U2OS cells (top panel) and COL1A1 knockout U2OS cells (bottom panel) (ab273846). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab138492 at 0.4μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Western blot - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

This data was developed using ab138492, the same antibody clone in a different buffer formulation. Different batches of ab138492 were tested on HFF-1 (human skin fibroblast) lysate at 0.5 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 220 kDa.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)This image is courtesy of an anonymous Abreview.

Paraffin-embedded human skin tissue stained for Collagen I using ab138492 at 1/3000 dilution in immunohistochemical analysis, followed by Goat anti rabbit Alexa Fluor^® 555.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
See Abreview
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)Image from Kishi Y et al. PLoS One. 2017;12(12):e0189522. Fig 2.; doi: 10.1371/journal.pone.0189522.

Type I collagen (ab138492) and Type III collagen immunostainings for control, Subtype I, and Subtype II adenomyotic cases.

The human type I collagen staining bands for adenomyotic cases were thicker than those of the control uteri, and were seen with more fine muscle bundles. Arrowheads indicate vascular walls. Original magnification: X100. Scale bar = 50μm.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

Immunohistochemistry of breast carcinoma staining Collagen I with ab138492 at 0.5μg/ml

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

IHC image of Collagen I staining in a section of frozen human cervix* performed on a Leica Biosystems BOND^® RX instrumen using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab138492, 0.01 ug/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

This data was developed using the same antibody clone in a different buffer formulation (ab138492).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

IHC image of Collagen I staining in human placenta formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval (EDTA based pH 9.0 solution, epitope retrieval solution 2) for 20 minutes. The section was then incubated with ab138492 at 1/1500, for 15 minutes 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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).
Immunohistochemistry (Frozen sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

IHC image of Collagen I staining in a section of frozen human uterus* performed on a Leica Biosystems BOND^® RX instrumen using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab138492, 0.05 ug/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

This data was developed using the same antibody clone in a different buffer formulation (ab138492).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach tissue labelling Collagen I with purified ab138492 at 1/1500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach tissue labelling Collagen I with unpurified ab138492 at 1/1500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

Formalin/PFA-fixed paraffin-embedded sections of human colon tissue staining Collagen I with unpurified ab138492 in immunohistochemical analysis.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

Formalin/PFA-fixed paraffin-embedded sections of human breast carcinoma tissue stained for Collagen I with unpurified ab138492 in immunohistochemical analysis.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
OI-RD Scanning - Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

Equilibrium disassociation constant (K_D)
Learn more about K_D

Click here to learn more about K_D
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138492).
Anti-Collagen I antibody [EPR7785] - BSA and Azide free (ab215969)

Western blot protocols
Immunohistochemistry protocols

Click here to view the general protocols

Datasheet download

Download

Publishing research using ab215969? Please let us know so that we can cite the reference in this datasheet.

ab215969 has been referenced in 4 publications.

Johnson BE et al. An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer. Cell Rep Med 3:100525 (2022). PubMed: 35243422
Sugali CK et al. The Canonical Wnt Signaling Pathway Inhibits the Glucocorticoid Receptor Signaling Pathway in the Trabecular Meshwork. Am J Pathol 191:1020-1035 (2021). PubMed: 33705750
Burlingame EA et al. Toward reproducible, scalable, and robust data analysis across multiplex tissue imaging platforms. Cell Rep Methods 1:N/A (2021). PubMed: 34485971
Chen Y et al. Circular RNA RSF1 promotes inflammatory and fibrotic phenotypes of irradiated hepatic stellate cell by modulating miR-146a-5p. J Cell Physiol 235:8270-8282 (2020). PubMed: 31960423

Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Switch on our new BETA site

Now available

In the coming months

Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Specificity

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Dissociation constant (KD)

Storage buffer

Carrier free

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Conjugation kits

Isotype control

Recombinant Protein

Related Products

Applications

The Abpromise guarantee

Target

Function

Tissue specificity

Involvement in disease

Sequence similarities

Post-translationalmodifications

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

Datasheet download

Certificate of Compliance

References (4)

Customer reviews and Q&As

Blocking abreview for Anti-Collagen I antibody [EPR7785] - BSA and Azide free

Dissociation constant (K_D)

Post-translational
modifications