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Detection of HuCAL derived 


Since HuCAL derived antibodies are human Fab molecules, they can be detected by antisera developed against human IgG F(ab’)2 preparations. In addition, HuCAL antibodies are usually equipped with one or two tags (genetically fused to the C-terminus of the antibody heavy chain), and can be detected by using commercially available anti-tag antibodies. We recommend the use of the following detection reagents:

  1. Detection of Fab scaffold
  2. Detection of C-terminal His6-tag (HHHHHH)
  3. Detection of C-terminal Myc-tag (EQKLISEEDL)
  4. Detection of C-terminal FLAG M2-tag (DYKDDDDK)
  5. Detection of C-terminal StrepII-tag (NWSHPQFEK)


Indirect ELISA

The detection of antigen immobilized on microtiter plates is the standard procedure used by AbyD for quality control of purified antibodies. The antigen is incubated in the well at a concentration of 5 μg/ml, and the purified HuCAL antibody (final concentration of 1 μg/ml) is used as primary antibody. Typically, Maxisorp ELISA plates from Nunc are used.

Indirect ELISA – Detection of Fab

CoatingAntigen 5μg/ml, o/n @ 4°CWash2 x PBST

(PBST buffer with 0.05 % Tween 20)Blocking5 % MP-PBST

(PBST containing 5 % milk powder)Wash2 x PBSTPrimary ABHuCAL antibody (2μg/ml) 1h @ RT in PBST or HiSpec BufferWash5 x PBSTSecondary ABα-Fab-AP conjugate1 (1:5 000 in HiSpec Buffer), 1h @ RTWash5 x PBSTDetectionAttoPhos2 (1:10 in H2O)

Indirect ELISA - Detection of c-terminal His6-tag

CoatingAntigen 5 μg/ml, o/n @ 4°CWash2 x PBST

(PBST buffer with 0.05 % Tween 20)Blocking5 % MP-PBST

(PBST containing 5 % milk powder)Wash2 x PBSTPrimary ABHuCAL antibody (2 μg/ml) 1h @ RT in PBST or HiSpec BufferWash5 x PBSTSecondary ABα-His6-HRP.

(1:400 in HiSpec Buffer), 1h @ RTWash5 x PBSTDetectionQuantaBlu Peroxidase Substrate, soluble

Indirect ELISA - Detection of c-terminal myc-tag

CoatingAntigen 5 μg/ml, o/n @ 4° CWash2 x PBST (PBS buffer with 0.05 % Tween 20)Blocking5 % MP-PBST (PBST containing 5 % milk powder)Wash2 x PBSTPrimary ABHuCAL antibody (2μg/ml) 1h @ RT in PBST or HiSpec BufferWash5 x PBSTSecondary ABα-c-myc-HRP5

(1:100 - 1:500 in HiSpec Buffer), 1h @ RTWash5 x PBST

Detection

QuantaBlu Peroxidase Substrate: Indirect ELISA – Detection of c-terminal FLAG M2-tag

CoatingAntigen 5 μg/ml, o/n @ 4°CWash2 x PBST

(PBS buffer with 0.05% Tween 20)Blocking5% MP-PBST

(PBST containing 5% milk powder)Wash2 x PBSTPrimary ABHuCAL antibody (2μg/ml) 1h @ RT in PBST or HiSpec BufferWash5 x PBSTSecondary ABα-FLAG M2-HRP7

(1:100 - 1:1000 in HiSpec Buffer), 1h @ RTWash5 x PBSTDetectionQuantaBlu Peroxidase Substrate (1:10 in H2O)

Indirect ELISA – Detection of c-terminal Strep-tagII

CoatingAntigen 5 μg/ml, o/n @ 4°CWash2 x PBST

(PBS buffer with 0.05% Tween 20)Blocking5% BSA-PBSTWash2 x PBSTPrimary ABHuCAL antibody (2μg/ml) 1h @ RT in PBST or HiSpec BufferWash5 x PBSTSecondary ABStrep-Tactin HRP conjugate

(1:4000 in 0.5% BSA-PBST), 1h @ RTWash5 x PBSTDetectionQuantaBlu Peroxidase Substrate.


Western Blot

The amount of antigen material loaded on the gel depends on the sensitivity of the detection system. For initial testing, use 200 μg of cell lysate proteins or 300ng of pure antigen. The purified HuCAL antibody should be used at a final concentration of 1 to 10 μg/ml. We recommend a starting concentration of 5 μg/ml.

For chemiluminescent detection, the system of choice is the use of an HRP conjugate in combination with a chemiluminescent substrate (e.g. ECL Plus, GE Healthcare). Alternatively, the BCIP/NBT substrate (AbD Serotec #BUF045A) can be used for alkaline phosphatase conjugates.

Western Blot - Detection of Fab

SDS-PAGEload 10ng-1μg antigen/laneBlottransfer to a PVDF membrane according to standard protocolsBlocking5 % MP-TBST (TBS containing 0.05 % Tween 20 and 5 % milk powder)WashRinse blot with TBSTPrimary ABHuCAL antibody (1 to 10μg/ml), 1 h @ RT in 1 % MP-TBSTWash3 x 5 min with generous amount of TBSTSecondary ABα-Fab-HRP conjugate

(1:5000 in 1 % MP-TBST), 1h @ RTWash3 x 5 min with generous amount of TBSTDetectionECL Plus chemilumenescence substrate

Western Blot - Detection of c-terminal His6-tag

SDS-PAGEload 10ng-1μg antigen/laneBlottransfer to a PVDF membrane according to standard protocolsBlocking5 % MP-TBST (TBS containing 0.05 % Tween 20 plus 5 % milk powder)WashRinse blot with TBSTPrimary ABHuCAL antibody (1 to 10μg/ml) 1h @ RT in 1 % MP-TBSTWash3 x 5 min with generous amount of TBSTSecondary ABα-His-HRP6-POD

(1:500 - 1:1000 in 1 % MP-TBST), 1h @ RTWash3 x 5 min with TBSTDetectionECL Plus chemilumenescence substrate

Western Blot - Detection of c-terminal myc-tag

SDS-PAGEload 10ng-1μg antigen/laneBlottransfer to a PVDF membrane according to standard protocolsBlocking5 % MP-TBST (TBS containing 0.05 % Tween 20 and 5 % milk powder)WashRinse blot with TBSTPrimary ABHuCAL antibody (1 to 10μg/ml), 1h @ RT in 1 % MP-TBSTWash3 x 5 min with generous amount of TBSTSecondary ABα-c-myc-HRP

(1:100 - 1:500 in 1 % MP-TBST), 1h @ RTWash3 x 5 min with generous amount of TBSTDetectionECL Plus chemilumenescence substrate

Western Blot – Detection of c-terminal FLAG M2-tag

SDS-Pageload 10ng-1μg antigen/laneBlottransfer to a PVDF membrane according to standard protocolsBlocking5% MP-TBST (TBS containing 0.05% Tween 20 and 5% milk powder)WashRinse blot with TBSTPrimary ABHuCAL antibody (1 to 10μg/ml), 1h @ RT 1 % MP-TBSTWash3 x 5 min with generous amount of TBSTSecondary ABα-FLAG M2-HRP

(1:100 - 1:1000 in 1 % MP-TBST), 1h @ RTWash3 x 5 min with generous amount of TBSTDetectionECL Plus chemilumenescence substrate

Western Blot – Detection of c-terminal Strep-tagII

SDS-Pageload 10ng-1μg antigen/laneBlottransfer to a PVDF membrane according to standard protocolsBlocking10% BSA-TBST, 1h @ RT (TBS buffer with 0.05% Tween 20 and 10% BSA)WashRinse blot in TBSTPrimary ABHuCAL antibody (1to 10μg/ml), 1h @ RT in 1% BSA-TBSTWash3 x 5 min with generous amount of TBSTSecondary ABStrep-Tactin HRP conjugate

(1:4000 in 1% BSA-TBST), 1h @RTWash3 x 5 min with generous amount of TBSTDetectionECL Plus chemilumenescence substrate