Protocols

Estimation of apparent rAB affinity by ELISA-based determination of EC50 and IC50

1.0 Introduction

Following expression and purification of soluble recombinant antibodies fragments (rABs), ELISA-based assays may be useful to verify reagent binding to target antigen. In general, two related methods are used to estimate rAB affinity but can also provide an indication of the optimal antibody concentration to be used in other applications and the specificity via direct comparison of binding to non-specific control proteins.

EC₅₀ or the concentration of antibody that gives half-maximal binding is determined by direct  and saturable binding of a rAB dilution series to both target antigen and a non-specific control protein. An estimate of affinity is interpreted from one-half the concentration at which rAB binding first achieves saturation. The assay can be done in a high-throughput manner using 3-5 fold dilutions of rAB of a stock antibody concentration in binding buffer. Since most antibodies will, at sufficiently high concentrations, begin to bind non-specifically to non-target proteins, this assay will enable determination of a [rAB] that provides maximal specific to non-specific signals when conducted using the appropriate non-specific control. EC₅₀ measurement allows for rank ordering of many unique clones, but may not provide an accurate measure of affinity.  It does however, allow for determination of a sub-saturating concentration of rAB that can provide for more accurate determination of rAB affinity in competitive ELISA.

A similar, yet more accurate determination of rAB affinity can be obtained through direct binding of rAB to immobilized target protein in the presence of series of concentrations of soluble target antigen in a competitive ELISA format.  The quantitation of rAB EC₅₀ is required to establish an accurate sub-saturating concentration of rAB for conducting the competitive binding experiment. The multipoint competitive ELISA then evaluates binding of the sub-saturating rAB concentration to immobilized antigen competed by pre-incubation with serial dilutions of antigen in solution to produce an inhibition curve from which the IC₅₀ value can be determined.

2.0 Overview Steps

• 2.1   rAB expressed and purified according to provided protocols 
• 2.2   rAB is serially diluted in buffer for use in binding assays
• 2.3   Following incubation with immobilized target and non-specific control proteins, binding of serially-diluted rAB to target is assessed using anti-Fab-HRP antibody which gives a A450 nm signal with TMB after reaction quenching
• 2.4   The rAB dilution that gives half-maximal binding is identified for subsequent use in competition experiments
• 2.5    The concentration of rAB that gives half-maximal binding is incubated with a range of concentrations of soluble antigen (typically 1 – 150 nM in multi-point, though single point competition can also be used to estimate affinity)
• 2.6    rAB in the presence and absence of soluble antigen is transferred to separate wells of a microplate to determine the percent inhibition elicited by known soluble antigen concentrations and to estimate affinity.  

Validation Criteria

We generally employ a 50 nM cut-off (IC50 as determined by multi-point competitive ELISA) in the evaluation of rABs to determine which of them will be further validated by immunoprecipitation and specificity testing.

3.0 Materials

Glass/plasticware

• Maxisorp 384 Immunosorp plates (Thermo, 12-565-347)
• Non-binding surface assay plates (Corning, 3641)

Reagents

• Anti-FLAG M2-HRP conjugate (Sigma, A8592)
• TMB Peroxidase substrate (KPL, 50-76-00)

Solutions

• Blocking buffer: 1X PBS pH 7.4 + 0.2% BSA
• PBT Binding Buffer: 1X PBS + 0.2% BSA + 0.05% Tween
• PT Wash Buffer: 1X PBS + 0.05% Tween

5.0 EC₅₀ determined by direct-binding ELISA

• 5.1    Immobilize antigen in a sufficient number of wells in a 384 well plate to generate a multi-point rAB dilution curve by incubating 30 μL of a 2 mg/mL solution in 1X PBS pH 7.4 overnight (O/N) with shaking at 4^oC.
• 5.2    Include an equivalent number of well coated with a non-specific control protein such as BSA, neutravidin, GST, or other appropriate control prepared in identical fashion.
• 5.3    Following immobilization, block plates by incubating with an excess of blocking buffer (1X PBS, 0.2% BSA) with shaking at room temperature (RT) for 1 hr.
• 5.4    Prepare a sufficient volume of a five-fold dilution series of purified rAB in PBT in a non-binding plate (generally 100 μL will be sufficient for each concentration of rAB) to test binding against both target and non-specific protein in a 384 well plate ranging from 1 to 150 nM.
• 5.5    Wash the antigen-coated plate 4X with PT buffer either by hand or using an automated plate washer. Remove excess buffer after wash complete by tapping the plate on paper towels.
• 5.6    Transfer 30 μL of diluted rAB to the antigen–coated plate and an equivalent number of non-specific control protein-coated plates and incubate for 1hr with shaking at 200 rpm at RT.
• 5.7    Wash eight times with PT buffer (This can be done with a plate washer using a slow dispense) and remove excess buffer after wash complete by tapping the plate on paper towels.
• 5.8    Add 30 μL of anti-FLAG M2 antibody fused to horseradish peroxidase (diluted 1:5000 in PBT buffer) to all wells including antigen and control protein-coated. Incubate 30 min with gentle shaking.
• 5.9    Wash six times with PT buffer and two times with PBS. Remove excess buffer after wash complete by tapping the plate on paper towels.
• 5.10    Add 30 μL of freshly prepared TMB substrate. Allow color to develop for 5–10 min or until blue colour develops.
• 5.11 Stop the reaction with 30 μL of 1.0 M H₃PO₄ and read spectrophotometrically at 450 nm in a microtiter plate reader.
• 5.12 The EC₅₀ is estimated by determining concentration of rAB that exhibits roughly one-half maximal absorbance from the point at which the rAB displays saturation behavior.  This can be done via curve fitting using fitting software such as SigmaPlot, GraphPad or Prism or by inspection to determine the OD at which saturation occurs, dividing by 2 and interpolating the concentration of rAB that results in this absorbance.

6.0 IC50 determined by multipoint competitive ELISA

• 6.1    Immobilize antigen in a sufficient number of wells in a 384 well plate to generate a multi-point rAB dilution curve by incubating 30 μL of a 2 mg/mL solution in 1X PBS pH 7.4 overnight (O/N) with shaking at 4^oC.
• 6.2    Following immobilization, block plates by incubating with an excess of blocking buffer (1X PBS, 0.2% BSA) with shaking at room temperature (RT) for 1 hr.
• 6.3    While blocking, prepare a mixture of purified rAB at the EC₅₀ in PBT with a range of [antigen] (typically from 0 – 150 nM) in non-binding plates as is described in Table 1.
• 6.4    Transfer 30 μL each rAB-antigen mixture in to separate wells of an antigen–coated plate and incubate for 30 min with shaking at 200 rpm at RT.
• 6.5    Wash eight times with PT buffer and remove excess buffer by tapping plates on paper towels.
• 6.6    Add 30 μL of anti-FLAG M2 antibody fused to horseradish peroxidase (diluted 1:5000 in PBT buffer). Incubate 30 min with gentle shaking.
• 6.7    Wash six times with PT buffer and two times with PBS and remove excess buffer by tapping plates on paper towels.
• 6.8 Add 30 μL of freshly prepared TMB substrate. Allow color to develop for 5–10 min or until blue colour develops.
• 6.9 Stop the reaction with 30 μL of 1.0 M H₃P_O4 and read spectrophotometrically at 450 nm in a microtiter plate reader.
• 6.10 The IC50 is estimated by determining concentration of antigen that results in roughly one-half maximal binding when compared to rAB binding in the absence of antigen.  This can be done via curve fitting using fitting software such as SigmaPlot, GraphPad or Prism or by inspection to determine the OD at which saturation occurs, dividing the maximal OD by 2 and interpolating the concentration of antigen that results in this absorbance.

Please send corrections, modifications and suggestions to smiersch@recombinant-antibodies.org

Antigen immunoprecipitation from spiked cell lysates by flow cytometry

1.0 Introduction / Description

Techniques involving immunoprecipitation (IP) are widely used in biomedical sciences. These include IP for protein interaction analysis such as IP-mass spectroscopy and chromatin IP (ChIP) methods for characterizing transcriptional regulation and epigenomics. Thus, we have made a particular effort in developing a method for identifying IP-positive antibodies. Whereas antibodies for IP have been conventionally validated using Western blotting, dot blotting, protein microarray and/or immunofluorescence, recent studies show that antibodies that are “passing” by these validation techniques often fail in IP experiments (1). A reason may be that conditions in which antibodies are used in IP are very different from those for the other methods: in IP, an antibody needs to efficiently capture an antigen, typically at a low concentration, in cell lysates. In Western blot, microarray and immunofluorescence, an antigen is separated, often concentrated and immobilized on solid support, which is detected with an antibody in a defined buffer. Consequently, distinct antibody properties are required to produce “passing” results for these two distinct types of procedures. In particular, antibody specificity and affinity are both critical factors in successful IP especially for capturing a low-abundance antigen from cell lysates. It should be noted that Western blot, microarray or immunofluorescence cannot directly assess antibody affinity.

We have recently developed an IP-based validation method that uses the same antibody–antigen orientation as in actual IP applications (2). We utilize flow cytometry that provide high sensitivity and high reproducibility. It revealed a very wide range of antibody affinities among commercial “ChIP-grade” antibodies to histone posttranslational modifications, which may explain why many of them fail in actual ChIP experiments. Furthermore, results from our IP assay substantially differed from those from peptide microarrays: microarrays gave false negative results (i.e. apparent cross-reactivity) for high-affinity antibodies and false positive results (i.e. apparent high specificity) for low-affinity antibodies. Although effective, a limitation of our IP method was that it required a series of closely related, purified antigens for assessing specificity. The “spiked IP” method examines whether or not an antibody of interest efficiently binds its cognate antigen, the same antigen used for antibody generation, in concentrated human cell lysates, instead of assessing specificity using a series of closely related antigens, making the IP validation more broadly applicable (3). Importantly, the use of high-concentration cell lysates in the assay does not interfere flow cytometry detection, and the assay is compatible with common solution conditions used in IP applications. Our current spike IP consists of two steps: (i) test IP efficiency with and without cell lysates with a single antigen concentration; (ii) optionally perform antigen titration for determining the dissociation constant (KD). The assay can be performed in two orientations: immobilized antibody or immobilized antigen. The protocol shown below is for the assay with an immobilized antibody, i.e. the format of actual IP. A high-specificity antibody should bind to its cognate antigen in cell lysates as well as in buffer. A reduction in binding signal by cell lysates indicate cross reactivity of the antibody with molecules in the lysates. This assay tests cross-reactivity with diverse types of molecules in cell lysates including proteins, nucleic acids, lipids and small molecules.

2.0 Overview steps

• 2.1 Dilute stock bead suspension
• 2.2 Aliquot diluted beads for test and controls
• 2.3 Add biotinylated recombinant antibody (rAB) to beads in control buffer and lysate and incubate
• 2.4 Transfer bead:rAB complex to a filter plate
• 2.5 Add 50 nM diluted antigen to filter plate containing bead:rAB complex and incubate
• 2.6 Wash beads in filter plate repeatedly
• 2.7 Add fluorescently-labeled streptavidin conjugate to beads and incubate
• 2.8 Wash beads in filter plate repeatedly
• 2.9 Resuspend beads in buffer for analysis
• 3.0 Analyze bead-bound rAB by flow cytometry

3.0 Materials

Glassware/Plasticware

• 96-well filter plate (MultiScreenHTS HV, 0.45μm pore size; Millipore)

Reagents

• Streptavidin-coated polystyrene beads (stock concentration is 6×105 beads/μl)
• (Bangs Lab Inc – CM01N)
• Streptavidin conjugated Phycoerythrin (Thermo)

Solutions

• High salt AFC buffer: 10 mM Tris-HCl, pH 7.9, 420 mM NaCl, 0.1% NP-40
• PBS/BSA: 20 mM Na₂HPO₄, pH 7.5, 150 mM NaCl, 0.1% BSA
• PBST/BSA: PBS/BSA + 0.1% Tween-20

4.0 Preparation of negative control beads

• 4.1 Dilute stock beads to 3 x103 beads / μl (1/200 dilution)
• 4.2 Resuspend diluted beads very well by vortexing.
• 4.3 You will need 10μl of diluted beads (3 x10³ beads / μl) per well, or 30 μL per antigen (measured in triplicate)
• 4.4 Aliquot appropriate amount of diluted beads into a new microfuge tube. Spin down briefly in refrigerated centrifuge: 9000 x g, 2 min, 4°C. Remove from centrifuge and discard the supernatant. Resuspend in PBST/BSA using the same volume as before.
• 4.5 For each antigen being tested, mix 30 μL of diluted beads with 3 μL of 50 μM biotin. Incubate for 15 min at 4°C with rotation.
• 4.6 For each antigen being tested, transfer 10μl of beads-biotin mixture into three wells on filter plate then vacuum.
• 4.7 Wash filter plate 3x with 200 μL of ice-cold PBST/BSA.

5.0 Preparation of  rAB-coated beads

• 5.1 Dilute stock beads to 3 x10³ beads/μl (1/200 dilution)
• 5.2 Resuspend diluted beads very well by vortexing.
• 5.3 You will need 10μL of diluted beads (3 x10³ beads/μl) per well, or 60 μL per antibody: 30 μL for triplicate positive binding measurements in buffer, 30 μL for triplicate positive binding measurements in lysate.
• 5.4 Aliquot appropriate amount of diluted beads into a new microfuge tube. Spin down briefly in refrigerated centrifuge: 9k x g, 2 min, 4°C. Remove from centrifuge and discard the supernatant Resuspend in PBST/BSA using the same volume as before.
• 5.5 For each rAB being tested, mix 60 μL of diluted beads with 60 μL of 10 nM rAB. Incubate for 30 min at 4°C with rotation.
• 5.6 Add 12 μL of 50 μM biotin for each rAB-bead mixture so as to block remaining biotin-binding sites of the streptavidin beads.
• 5.7 Incubate for 15 min at 4°C with rotation.
• 5.8 Transfer 20 μL of beads-rAB-biotin mixture into six wells on filter plate then vacuum. This should be done in parallel with step 4.6.
• 5.9 Wash filter plate 3x with 200 μL of ice-cold PBST/BSA.
• 5.10 Remove solution by vacuum and proceed to target incubation.

6.0 Antigen Incubation

• 6.1 Prepare 120 µL of 50 nM biotinylated antigen in buffer and 60 µL of 50 nM antigen in 3 OD HEK293 lysate.
• 6.2 Dispense 20 µL of antigen in buffer into three of the filter plate wells with the corresponding rAB coated beads and 20 µl of antigen in lysate into rest of the filter plate wells with the corresponding rAB beads.
• 6.3 Dispense 20 µL of antigen in buffer to into three of the filter plate wells with negative control beads.
• 6.4 Incubate for 30 minutes at 4°C with shaking.

7.0 Staining

• 7.1 Add 20 uL of 1/100 SAV-PE (Thermo) in PBS/BSA to beads.            
• 7.2 Incubate sample for 30 min at 4°C on plate shaker.
• 7.3 Wash filter plate 3x with 200 μL of ice-cold PBST/BSA.

8.0 Flow Cytometry Measurements

• 8.1 Resuspend beads in 100 μL of PBST/BSA.
• 8.2 Measure with Intellicyt. 10 s sip time, FL2 height. (this step depends on the instrument to be used).
  NOTE – For titration, experiments are performed with a range of antigen concentration.

Please send corrections, modifications and suggestions to smiersch@recombinant-antibodies.org

Immunofluorescent localization of antigens in selected cell lines

1.0 Introduction / Description

rABs generated by the RAN group were obtained by selection against isolated transcription factor domains.  As one component of our validation pipeline, the ability of each rAB to bind to it’s cognate full length, transcription factor in a cellular environment was evaluated by immunofluorescence assay in native cell lines that endogenously express varying amounts of the target transcription factor using the following protocol:

2.0 Overview steps

• 2.1 Deposit cells at a density suitable for staining and imaging
• 2.2 Fix cells by treatment with ice-cold methanol
• 2.3 Permeabilize cells by treatment with 0.25%Triton X-100
• 2.4 Block cells by 1% serum treatment
• 2.5 Stain cells with primary antibody and wash
• 2.6 Stain cells with secondary antibody and Hoechst dye and wash
• 2.7 Image not less than 100 cells using Opera high-throughput imager (Perkin Elmer) and quantitate immunofluorescence intensity in comparison to cells stained with secondary antibody alone

3.0 Materials

Glassware/Plasticware

• Perkin Elmer tissue culture treated CellCarrier plates (product #:6005550)

Reagents

• Goat serum (G9023-10mL)
• Secondary Fluorescent conjugated antibody (Human anti-rAB FITC conjugate, Jackson Labs (#109-546-097))
• Hoeschst 33342 Dye (Lifetech, H3570)

Cell lines

• The following cell lines have were used to assess endogenous expression and localization:
• ES-2, JHOC-9, A2780Cis, 3345A, A2780, HPAFII, HepG2, 293T

4.0  Cell deposition – Day 1

• 4.1 Seed cells at 5,000-10,000 cells per well
• 4.2 Swirl gently to distribute cells evenly, then incubate overnight at 37°C to allow adherence.

5.0 Cell permeabilization and fixation – Day 2

• 5.1 Remove media from wells and wash once with 1x PBS
• 5.2 Add 100 ml of 70% -20 °C ice cold Methanol (prepared with MilliQ) water per well        
• 5.3 Place in -20 °C freezer and incubate for 10 minutes
• 5.4 Rinse 3x with PBS for 5min each
• 5.5 Add 100ml of 0.25% Triton X-100/PBS to each well 
• 5.6 Incubate for 10 minutes at RT
• 5.7 Wash 3x with PBS for 5min each
• 5.8 At this point, cells can be stored for several days in PBS solution at 4 °C before staining

6.0 Blocking and immunostaining of non-specific binding sites on cell samples

• 6.1 Block in 1% normal goat serum/PBS for 1 hour at RT or overnight at 4 °C
• 6.2 Remove blocking solution
• 6.3 Add 100 μL of primary antibody diluted in 1% goat serum/PBS or negative control (goat serum/PBS alone)
• 6.4 Incubate 1 hour at RT
• 6.5 Wash 3x with PBS 5min each
• 6.6 Add 100 μL secondary antibody diluted in 1% goat serum/PBS with 2 mg/mL Hoechst dye
• 6.7 Incubate 1 hour at RT in the dark, and keep coverslips in the dark as much as possible from this step on.
• 6.8 Wash 3x with PBS 5min each.
• 6.9 For storage of stained cells in 96 well plates, add 100 mL of PBS and cover with aluminum foil seal and leave up to 5 days.

7.0 Quantification and data format

• 7.1 Image immunostained cells on an imaging system capable of handling 96 well plates at an appropriate and constant incident light, gain and sensitivity setting.
• 7.2 Manually inspect images to determine apparent localization of immunostained target transcription factor in various cell lines.
• 7.3 Categorization of fluorescence localization in the cell was indicated using five simple GO-based descriptors: cytoplasmic, nuclear, mixed cytoplasmic/nuclear, other, miscellaneous, or not tested.

Please send corrections, modifications and suggestions to smiersch@recombinant-antibodies.org.

Antigen immunoprecipitation for mass spectrometric analysis

1.0 Introduction / Description

As one component of the validation pipeline, the ability of each rAB to bind to its cognate antigen in a cellular environment will be evaluated by immunoprecipitation assay in native cell lines known to express the target protein. Mass spectrometric analysis of immunoprecipitated antigen will provide measurement of target peptide spectral counts and the relative amounts of IP’d target, the pull-down of related proteins (thus providing an indication of specificity) and the pull-down of other target-bound proteins (thus providing additional data of potential biological significance).

2.0 Overview steps

• 2.1 Culture cells expressing the target of interest
• 2.2 Collect cells in lysis buffer, lyse by sonication and collect lysates
• 2.3 Form the antibody-target complex by rAb incubation in lysate
• 2.4 Immunoprecipitate protein complexes with streptavidin beads
• 2.5 Wash beads, elute and flash freeze immunoprecipitated material
• 2.6 Trypsinize protein complexes and extract/desalt peptides for MS analysis

3.0 Materials

Glassware/Plasticware/ Instrumentation

• 10 cm tissue culture dishes
• Zip tips (Millipore catalogue # ZTC18M960)
• Mass Spec Plate (Axygen scientific from VWR – Cat#PCR-96-FS-C

Reagents

• Tris-HCl (SigmaT5941-1KG)
• NaCl
• NP-40 (Pierce / Thermo, Cat #28324)
• Protease/phosphatase inhibitors (Pierce/Thermo, Cat #78440)
• benzonase (Sigma, Cat #E1014-5KU)
• streptavidin magnetic dynabeads (used for biotinylated rAbs) (Invitrogen, Cat #65602)
• anti-FLAG M2 magnetic beads (used for FLAG-tagged rAbs) (Invitrogen, cat#MM8823)
• Protein A magnetic dynabeads (used for mAbs) (Invitrogen, Cat # 10001D)
• Protein G magnetic dynabeads (used for mAbs) (Invitrogen, Cat # 10003D)

3.3 Solutions

• AFC  (High Salt) (Lysis buffer)
   Final concentrationStockFor 40 mL 10X bufferTris-HCl, pH7.910 mM2 M2 mLNaCl420 mM5 M33.6 mLNP-400.1%10%4 mLH2O  0.4 mLAdd protease inhibitor just before use.
  Add 1 mM Ni to the buffer for demethylases.
• FC  (Low Salt) (Dialysis/Wash buffer)
   Final concentrationStockFor 40 mL 10X bufferTris-HCl, pH7.910 mM2 M2 mLNaCl100 mM5 M8 mLNP-400.1%10%4 mLH2O  26 mLAdd protease inhibitors just before use, same as high salt.
• Wetting and Equilibration solution70% CAN in 0.1% FA
• Washing solution 100% H₂0 in 0.1% FA

4.0 Preparing cell lysate (HEK293 cells)

NOTE: Use non-autoclaved eppendroff tubes and tips!

• 4.1 Everything on ice or at 4 ^oC unless indicated otherwise.
• 4.2 Thaw frozen cell pellets immediately with 1X High Salt AFC buffer  (1 mL per plate) with protease and phosphatase inhibitors
• 4.3 Perform 3 freeze-thaw cycles by moving samples between ethanol/dry ice and 37 ^oC water bath, mixing frequently to prevent temperature from reaching above 4 ^oC
• 4.4 Sonicate 5 times, 0.3 s on/0.7 s off (per 1 ml), (8 per 2 ml, 12 per 5 ml)
• 4.5 Incubate for 30min at 4 ^oC with benzonase nuclease to remove RNA and DNA (to the final concentration of 12.5-25 units/mL).
• 4.6 Centrifuge at 13,000 rpm for 30 min at 4 ^oC.  Discard pellet and retain supernatant. 
• 4.7 Measure protein concentration (nanodrop), usually 7-10 mg/mL

5.0 Antibody binding

• 5.1 Use 3-5 mg per IP. Add ~2 μg of antibody into the lysates and incubate at 4 ^oC overnight (depending on the stability of the proteins), 2hrs incubation is sufficient for most antibodies.
• 5.2 Wash with high salt AFC buffer 20 mL of magnetic beads per IP, 2x.
• (NOTE: The beads are different for different types of antibodies; flag-tag rABs – use magnetic beads from SIGMA; avi-tagged rABs use streptavidin dynabeads from Life Technologies; IgGs- use protein A/G dynabeads from Life Technologies)
• 5.3 After last wash resuspend in 20 mL of low salt AFC buffer and add 20 mL to the lysates with antibodies. 
• 5.4 Incubate for 2-4 hr at 4 ^oC.
• 5.5 Wash the beads with 1 ml of 1x low salt lysis buffer 3 times at 4 ^oC for 5 -10 min and 2x with low salt lysis buffer without detergent.
• FOR IP-WB we just elute with protein loading buffer (60 microliters) and load 30 mL on protein gel.
• 5.6 Elute 4×50 mL of 0.5 M ammonium hydroxide, flash freeze in liquid nitrogen
• 5.7 Dry and perform trypsin digest according to protocol below.

6.0 Trypsin digestion

NOTE: All the steps should be done in HPLC grade water or 50mM NH₄HCO₃
NOTE: Use non-autoclaved tubes and tips
NOTE: Make sure you work in a clean environment to prevent keratins and polymer contamination, hood is not necessary but careful work environment is essential!

• 6.1 Dry NH₄OH samples in the speed vac (about 1 – 1.5 hrs)
• 6.2 Reconstitute in 44 mL of 50 mM NH₄HCO₃
• 6.3 Add 1 mL of 100 mM TCEP-HCL . Shake at 37 ^oC for 1 hr
• 6.4 Cool to room temperature and add 1 mL 500 mM iodoacetamide. Shake at room temperature and in dark for 45 mins
• 6.5 Add 1 mg Trypsin. Shake overnight at 37 ^oC
• 6.6 Add 2 mL acetic acid to stop the reaction
• 6.7 Store at 4 ^oC
• 6.8 The volume is 50.5 mL

7.0 Desalting Protocol (for Zip-Tip)

NOTE: The maximum volume for Zip Tip is 10 ml

• 7.1 Equilibration:
  • 7.1.1 Aspirate 10 mL Wetting and Equilibration solution into zip-tip
  • 7.1.2 Dispense waste
  • 7.1.3 Repeat twice
  • 7.1.4 Aspirate 10 mL with washing solution
  • 7.1.5 Dispense waste
  • 7.1.6 Repeat twice
• 7.2 Binding and washing:
  • 7.2.1 Aspirate 10 mL and dispense into the sample, repeat at least 20 times so all the peptides are bound to the tip (do not throw away the samples – just pipette up and down)
  • 7.2.2 Aspirate 10 mL of washing solution twice and dispense
• 7.3 Elution:
  • 7.3.1 Dispense 10 mL of Wetting and Equilibrating solution into 96 well mass spec plate four times (Final sample volume is 40 mL)
  • 7.3.2 Dry the samples
• 7.4 Perform mass spec analysis

Please send corrections, modifications and suggestions to smiersch@recombinant-antibodies.org.