Biophysical/Structural Biology Technologies & Requirements

X-ray of apo Antibody

Contact: Joel Harp

e-mail: joel.harp@vanderbilt.edu

phone: (615)936-3885

Amount of Material: 1-5mg concentrated to >= 10 mg/mL

Purity of Material: >95%

Comments: Provide in low ionic strength buffer, complete sequence, may require additional material for optimization, concentration depends on solubility and has ranged from 10 mg/mL to 44.5 mg/mL

X-ray of Antibody Complex

Contact: Amritraj Patra

e-mail: amritraj.patra@vanderbilt.edu

phone: (615)600-1596

Amount of Material: 1-5mg of antibody-antigen complex

Purity of Material: Purified by size exclusion column and concentrated to ~10 mg/mL

Comments: Provide in low ionic strength buffer, complete sequence, may require additional material for optimization, concentration depends on solubility and has ranged from 10 mg/mL to 44.5 mg/mL

HD/X of apo Antibody

Contact: Marcus Nagel

e-mail: mbmnagel@gmail.com

phone: (615)569-8861

Amount of Material: 5umol+ (or 250ug+)

Purity of Material: 50% +

Comments: Detailed information of mature sequence and PTMs, preferably little glycosylation, no aggregates, no detergents, ideal concentration is around 2mg/mL

HD/X of Antibody Complex

Contact: Marcus Nagel

e-mail: mbmnagel@gmail.com

phone: (615)569-8861

Amount of Material: each 5umol+ (or 500ug+)

Purity of Material: 50% +

Comments: Detailed information of mature sequence and PTMs, preferably little glycosylation, no aggregates, no detergents, ideal concentration is around 2mg/mL

Cross-linking of Antibody Complex

Contact: Marcus Nagel

e-mail: mbmnagel@gmail.com

phone: (615)569-8861

Amount of Material: 15ug +

Purity of Material: 30% +

Comments: Detailed information of mature sequence and PTMs, preferably little glycosylation, AB and Antigen can be premixed, preferably amine-free buffer

ITC of Antibody Complex

Contact: Alberto Cisneros

e-mail: alberto.cisneros@vanderbilt.edu

phone: (512)635-7261

Amount of Material: Cell sample 5-20 uM, 300uL – between 100ug and 1mg depending on molecular weight of antigen; Syringe concentration 50-200uM – 180 to 700ug for Fab, 525ug to 2.1mg for IgG

Purity of Material:

Comments: Materials must undergo buffer exchange and must be as pure as possible

EM of Antibody Complex

Contact: Amritraj Patra

e-mail: amritraj.patra@vanderbilt.edu

phone: (615)600-1596

Amount of Material: 50-100 uL of ~1 mg/mL of sample in commonly used biological buffer

Purity of Material: Ideally sample should be as homogeneous as possible

Comments: Some sample buffers may cause problems, including presence of detergent, phosphate, reducing agent or glycerol

Octett of Antibody Complex

Contact: Alex Sevy

e-mail: alexander.m.sevy@vanderbilt.edu

phone: (615)343-8064

Amount of Material: 20-100 uL each of antibody and antigen

Purity of Material: >90%

Comments: Ideally the antigen should be biotinylated.  Amount of protein needed is highly dependent on affinity.  Accurate KD can only be determined for antibodies with high off rate

Wasatch SPR of Antibody Complex

Contact: Alex Sevy

e-mail: alexander.m.sevy@vanderbilt.edu

phone: (615)343-8064

Amount of Material: 20-100 uL each of antibody and antigen

Purity of Material: >90%

Comments: Can be done label-free. Amount of protein needed is highly dependent on affinity.

SAXS of Antibody Complex

Contact: Clayton Wandishin

e-mail: clayton.m.wandishin@vanderbilt.edu

phone: (703)568-1944

Amount of Material: 24uL of sample at 1 mg/mL, 24uL of sample at 3 mg/mL, 24uL of sample at 5 mg/mL, 48uL of sample buffer (2 x 24uL wells).  This comes out to 216ug total for each sample

Purity of Material: >95% with either SEC or dialysis preparation

Comments: Buffer needs to be EXACT match, not an equivalent, and must contain more than 1M salt with <200mM preferred.  2% glycerol is also recommended.