Cell Wall Antibodies

Cell wall antibodies for polysaccharide research

Paul Knox Antibody Collection

The JIM and LM series of antibodies are a substantial resource for detailed analysis of plant cell wall carbohydrates. These antibodies are monoclonal, typically derived from rats, and are highly regarded for their sensitivity and specificity as molecular probes. This amazing collection of probes was developed and maintained by Paul Knox at the University of Leeds up until his retirement. Use the above button to navigate to Pauls websitse

Their versatility allows use in various analytical techniques, including microscopy and quantitative assessments of cell wall components across a wide spectrum of plant materials. Our main uses are immuonolabelling of plant sections, and ELISA of extracted cell wall components, and we often carry out collaborative work using these techniques - to discuss this get in touch using the contact section.

Some antibodies are better characterised than others so have greater detail about them available

Detailed Antibody Binding notes

General considerations

Critical for plant cell wall research is that glycan-directed antibodies are primarily epitope-specific rather than strictly polymer-specific.17 This distinction is crucial because the complex and diverse nature of plant cell wall glycans means that a particular epitope might be present on more than one class of wall polymers, especially for structures containing arabinose and/or galactose. E.g. while JIM5/JIM7 has "no known cross-reactivity with other polymers", other antibodies like LM5 and LM6m may exhibit binding to AGPs in addition to their primary galactans and arabinan targets. Similarly, LM12 can bind to both feruloylated pectin and feruloylated xylan, illustrating how a single epitope can be found in different polysaccharide contexts.

The dynamic nature of plant cell walls further complicates interpretation, as glycan epitopes can vary significantly between different tissues, organs, and even species. Studies consistently show developmental regulation of these epitopes, meaning their presence and presentation can change throughout a plant's life cycle. Some antibodies, such as LM8 and LM9, demonstrate highly restricted reactivity, binding only to specific tissues or plant families. Therefore, relying solely on broad reactivity assumptions can lead to misinterpretations.

A crucial aspect of interpretation is understanding that the absence of labeling cannot be unambiguously interpreted as the absence of the epitope or the corresponding polymer. The epitope might be present but in a modified form, masked, or in a concentration below the detection limit of the antibody

Controls

The implementation of immunological controls is important for accurate data interpretation. These controls help to distinguish specific antibody binding from non-specific signals or background noise. Although all controls are not always possible the following are useful controls to include:

Sections incubated solely with buffer to assess autofluorescence.
Sections omitting primary antibody, to assess non-specific binding of the secondary antibody.
Incubation of sections with an isotype-matched antibody from the same host species (rat, in this case) that does not bind to plant tissues. This control helps to confirm that any observed signal is due to specific antigen recognition by the primary antibody, rather than non-specific interactions related to the antibody's isotype or host origin.
Pre-incubation of the primary antibody with a known ligand or purified epitope. If the labelling is abolished or significantly reduced, it confirms the specificity of the antibody-epitope interaction.

Non reducing end:

Refers to the end of a polysaccharide chain that does not possess a free anomeric carbon capable of undergoing oxidation. This is in contrast to the "reducing end," which does have such a group. Polysaccharides are long chains of sugar units: one end is the "start" and the other is the "end." These ends have distinct properties. The non-reducing end is often where new sugar units are added during synthesis, or where enzymes might initiate degradation.

When an antibody binds specifically to the NRE (Non-Reducing End) of a polysaccharide, it means the epitope that the antibody recognizes is located precisely at the very end of a polysaccharide chain that lacks a reducing group. This often indicates the presence of a terminal sugar residue or a specific short sequence that is only exposed at the non-reducing end of the polymer.

Such antibodies are incredibly valuable for:

- Detecting Polymer Chain Breaks: If a polysaccharide chain is cleaved, new NREs are created. An NRE-specific antibody can therefore indicate sites of degradation or processing.
- Identifying Specific Polymer Architectures: Some polysaccharides are highly branched, and certain branches might terminate with specific NREs. An NRE-specific antibody can help elucidate these branching patterns.
- Studying Biosynthesis and Turnover: These antibodies can provide insights into enzymes that add or remove sugar units from the non-reducing end, or enzymes that cleave polymers to create new NREs.

Hemicelluloses

Xylan

LM10

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Non-reducing end (NRE) of xylans / NRE (1→4)-β-D-xylan

Notes: Distinct from LM11 in its binding, specifically recognizing the non-reducing end (NRE) of xylans, or NRE (1→4)-β-D-xylan. This NRE specificity is crucial for understanding xylan architecture and modification.

LM11

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: (1→4)-β-D-xylan / arabinoxylan

Notes: Core probe for heteroxylan. Binds to 1,4-xylosyl residues, specifically (1→4)-β-D-xylan and arabinoxylan

LM12

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Ferulic acid, feruloylated pectin / feruloylated xylan

Notes: Can bind to feruloylated heteroxylan (when associated with pectin) or feruloylated pectin (when associated with xylan).

LM27

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Unknown epitope assoc. grass xylan

Notes:

LM28

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Glucuronosyl residues / glucuronoxylan

Notes: Targets glucuronosyl residues, characteristic of glucuronoxylan.

Xyloglucan

LM15

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: XXXG motif of xyloglucan

Notes: Core probe for heteroxylan.

LM24

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Galactosylated xyloglucan

Notes:

LM25

Species: Rat, Isotype: , Recomended Dilution: (1:30)

Epitope: XXXG/galactosylated xyloglucan

Notes: High affinity; recommended for xyloglucan detection.

Other

LM21

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Heteromannan

Notes:

LM22

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Heteromannan

Notes:

Callose

10H2

7H10

1A11

Pectins -HGA

For pectic HG we recommend the combined use of LM19 (unesterified HG) and LM20 for high methylesterified HG. JIM7 is also always additionally recommended for any analysis as it binds widely to pectin.

JIM5

Species: Rat, Isotype: IgG, Recomended Dilution: TBD (1:10)

Epitope: Partially methyl-esterified & unesterified homogalacturonan (α-1,4-linked HG)

Notes: a well-characterized monoclonal antibody that specifically recognizes partially methyl-esterified and unesterified epitopes of homogalacturonan (HG), which is the α-1,4-linked homogalacturonan domain. This antibody exhibits no known cross-reactivity with other polymers. In terms of performance, JIM5 has been tested and is effective in Immunofluorescence and ELISA applications, with a recommended dilution of 1:10 for both. Polygalacturonic Acid serves as a reliable positive control for assays involving JIM5.

JIM7

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Partially methyl-esterified homogalacturonan (highly methyl-esterified HG, up to 80%)

Notes: Targets homogalacturonan and specifically recognizing highly methyl-esterified HG epitopes, which can include up to 80% methyl-esterified residues. Due to its broad binding to pectin, JIM7 is consistently recommended for general pectic HG analysis and as a positive labelling control. It has been extensively utilized in studies to investigate the developmental regulation of HG epitopes, particularly during processes like somatic embryogenesis.8

LM7

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Partially Me-HG / Non-blockwise de-esterified HG epitope.

Notes: specifically targeting non-blockwise de-esterified HG.

LM8

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Xylogalacturonan

Notes: May be restricted in tissue/species range; labels xylogalacturonan at sites of cell detachment/separation

LM18

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Partially Me-HG / no ester; low methyl-esterified HG

Notes:

LM19

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Unesterified HG (Homogalacturonan) / partially Me-HG / no ester

Notes: A key antibody that recognizes unesterified homogalacturonan (HG) and partially methyl-esterified HG with no ester groups. This antibody has been effectively employed in immunolabeling studies to detect low methyl-esterified HG epitopes. Recommended for combined use with LM20 for pectic HG analysis

LM20

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: High methylesterified HG / partially Me-HG

Notes: Complements LM19 by binding to high methylester HG and partially methyl-esterified HG. The combined application of LM19 and LM20 is a standard practice for a thorough investigation of pectic HG, allowing researchers to differentiate between various degrees of methyl-esterification. LM20 has been instrumental in studies examining developmental regulation of highly methyl-esterified HG epitopes.

Pectins - non-HGA

LM5

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: NRE (1→4)-β-D-galactan

Notes: May also bind to AGPs.

LM6-M

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: (1→5)-α-L-arabinan

Notes: May also bind to AGPs.

LM9

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Feruloylated (1→4)-β-D-galactan

Notes: Binds to epitope specific to Amaranthaceae (e.g., sugar beet, spinach)

LM13

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Linearised (1→5)-α-L-arabinan

Notes:

LM16

Species: Rat, Isotype: IgM, Recomended Dilution: TBD (1:10)

Epitope: RG-I epitope associated with arabinans

Notes: Its binding may be sensitive to galactosidase action, indicating that galactosyl residue(s) on the rhamnogalacturonan backbone might be part of its epitope. Furthermore, LM16 recognizes an epitope associated with arabinans, which can be generated by arabinofuranosidase action and the removal of arabinosyl residues.

LM26

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Branched (1,6-Gal) (1→4)-β-D-galactan

Notes:

AGPs

It can be hard to predict which AGP MAb to select as these glycan epitopes vary between tissues, organs and species. JIM13 and LM2 are a good place to start as they usually detect something in a section or an extract. Do not forget that a single AGP glycan epitope is unlikely to detect all AGPs in an organ.

JIM4

Species: Rat, Isotype: Unknown.

Epitope: Arabinogalactan protein

JIM13

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: AGP-glycan

Notes: Recognises an epitope found on Arabinogalactan-Proteins (AGPs). For initiating work on AGPs, JIM13 is often suggested as a starting point because it usually yields detectable signals.

JIM14

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: AGP-glycan

Notes:

JIM15

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: AGP-glycan

Notes:

JIM16

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: AGP-glycan

Notes:

LM2

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: β-linked-GlcA in AGP glycan

Notes: recognizes β-linked-GlcA within AGP glycans. Similar to JIM13, LM2 is considered a good starting point for AGP MAb selection due to its consistent ability to detect epitopes in sections or extracts

LM14

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: GlcA in AGP glycan

Notes:

LM30

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: AGP-glycan

Notes:

Extensins

For starting with extensins we suggest the use of LM1 and JIM20.

JIM11

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Extensin

Notes:

JIM12

Species: Rat, Isotype: IgM , Recomended Dilution: TBD (1:10)

Epitope: Extensin

Notes:

JIM19

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Extensin

Notes:

JIM20

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Extensin

Notes:

LM1

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Extensin

Notes: targets extensin, a key glycoprotein in the plant cell wall. It is suggested for initial investigations into extensins, complementing the JIM series extensin antibodies.

Others

JIM18

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Glyco-phospholipid, membranes

Notes: we don't have this antibody currently

LM4

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Pea amine oxidase cell walls

Notes: we don't have this antibody currently

LM23

Species: Rat, Isotype: , Recomended Dilution: TBD (1:10)

Epitope: Non-acetylated xylosyl in xylogalacturonan, xylan, fucoidan preps

Notes:

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