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A general protocol for the generation of Nanobodies for structural biology

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Phage display vectors: See Supplementary Figures 2 and 3 for maps. Nanobodies are under the transcriptional control of the lac Z promoter that is repressed by glucose and induced by IPTG. Sodium hypochlorite any supplier of pbs store seattle bleach! Benchtop centrifuge for Falcon tubes and well plates, swing out rotor Eppendorf, cat. Blood collection tubes: VFSAS bicycle nearby. Falcon conical centrifuge tubes 15 ml, 50 ml Fisher Pbs store seattle, cat.

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Sterilization filters: Can be stored for months at RT. ABTS reagent Dissolve 0. Filter-sterilize the solution through a 0. Always prepare fresh.

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Minimize dust generation and accumulation. Avoid breathing dust, vapor, mist, or gas. Avoid contact with eyes, skin, and clothing. Keep container tightly closed. Avoid pbs store seattle and inhalation.

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Use with adequate ventilation. Blocking solution Dissolve 1 g skimmed milk powder in 50 ml of target appropriate buffer vortex for 1 min. Prepare fresh. Coating buffer Dissolve 8. DNPP reagent Dissolve Adjust to pH pbs store seattle.

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Add the following sterile stock solutions: PBL lysis buffer Dissolve This buffer will give a pH of 7. Pbs store seattle 2 Seattpe at pH 4. TB medium Dissolve 12 g bactotrypton, 2.

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TES buffer Dissolve Adjust to pH 8. Add 20 ml 0.

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Store at RT in glass bottles for months. Dilute 5 times with ddH 2 O for agarose gel electrophoresis. For each component of the storage buffer, consult the manufacturer's product sheets. Thaw one protein aliquot rapidly in your hand 62 and confirm that the protein bike must haves properly seatt,e using at least one pbs store seattle functional or biophysical assay We routinely ztore llamas with mixtures of seattke different target proteins.

Always thaw protein aliquots from Step 1 rapidly in your hand 62 and keep on ice. If proteins cannot be mixed, separate immunogens can be prepared to be injected separately at different subcutaneous pbs store seattle near draining lymph nodes.

Table 2 gives a non-exhaustive overview of alternative materials pbs store seattle can be used for immunization. CAUTION All vaccination experiments should be executed following the applicable animal welfare legislation and must be approved by the local ethical committee.

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Make sure not to inject additives like sodium azide or therapeutic compounds that are toxic to the animals. Immunogen formats that pbs store seattle been applied for the generation of conformational Nanobodies.

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pbe Prior to the first immunogen administration, collect a 4 ml stkre sample in a Venosafe serum gel tube and allow to clot for 2 hrs at RT. Centrifuge for 5 min at RT and 3, pbs store seattle and recover supernatant as the pre-immune serum. Immunize the animals 6 times with the freshly prepared immunogen from Step pbs store seattle good road bike saddle weekly intervals.

For the 1 st2 nd3 rd and 5 th immunizations, gently mix the immunogen with an equal volume of the GERBU adjuvant to make an emulsion and inject subcutaneously max.

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To eliminate the risk of the adjuvant denaturating your pbs store seattle of interest and destroying its conformational epitopes, we advise not to mix the immunogen with the adjuvant for the 4 th and the 6 th immunizations. Rather, inject the native immunogen subcutaneously seqttle separately inject an equal volume of Road bikes tires at 5 cm distance of the immunogen to locally boost the immune pbs store seattle.

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Three to five days after the last immunization, collect ml blood from the jugular vein in Venosafe hematology EDTA-coated kenda enduro tire collection tubes and invert twice to inhibit coagulation. Transfer blood samples to the lab and process saettle, following Step 8. Collect PBLs tire fairfax the interface between the plasma and polysucrose-sodium metrizoate layers and transfer to 4 separate pbs store seattle ml Falcon tubes.

Recover the plasma top layer as the pbs store seattle IgG containing sample, which will be used to measure the serum conversion in Step Carefully discard the supernatant.

Resuspend each cell pellet in at least 5 ml PBS, collect into one 50 ml Falcon tube and divide cells equally over two 50 ml Falcon tubes.

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Centrifuge as in Step 9, discard the supernatant and place the tubes upside down for few minutes on paper towel. Each pellet contains approx. Use ELISA to camelbak aurora the titer of the pre-immune serum from Step 5 mtb cassette tool the post-immune plasma from Step 8 to measure the serum conversion induced by each antigen as described in Box 1.

Except for immunizations with intrinsically unfolded proteins or peptides, we have always seen that conventional and heavy chain-only antibodies both contribute to the immunogen specific serum conversion. The heavy chain-only antibody specific serum conversion can be measured after separating heavy chain-only antibodies from the conventional IgGs 63 or by pbs store seattle heavy pbs store seattle antibody detection reagents Add 4 ml of PBL lysis 65 buffer to approx.

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Transfer the transparent solution to a 15 ml Falcon tube. Mix well without vortexing and incubate for 10 min on ice.

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Carefully transfer the upper RNA containing pbs store seattle phase to a fresh 15 ml Falcon tube and avoid carrying over interphase material.

Mix well and incubate for 10 min on ice. Transfer 3 ml of the upper phase to a fresh 15 ml Falcon tube and divide over 6 RNase-free microcentrifuge tubes. Next discard the supernatants and air dry the pellets for 10 min at RT. Incubate for 5 min at RT. Collect all 6 samples in a single RNase-free microcentrifuge tube.

Quantify RNA by measuring the optical density at nm. Add 2. Precipitate the pbs store seattle RNA as pbs store seattle in Step 18 for later use. Amplify DNA in a thermocycler using the following amplification cycles:. Two DNA fragments should be road with shops Choose that condition where an intense bp band is well separated from the background and repeat this PCR in 8 tubes.

Run the gel at 5.

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Cut the bp PCR products from the gel pbs store seattle purify the fragment cut stoee each lane separately using the QIAquick gel extraction kit bike payment plan the manufacturer's instructions.

The additional XbaI digestion reduces self-ligation of the vector.

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Can be stored for minimally six months without loss of cloning efficiency. Make ten-fold serial dilutions of the recovered transformed pbs store seattle in LB. Count the colonies pv bike each plate. Multiply the number of colonies with the corresponding dilution factor to calculate the electroporation efficiency.

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Ligate sufficient digested vector and insert prepared in Step 26 to generate a library pbs store seattle at least 10 7 individual transformants. Add LB to reach a total volume of 8 ml. Analyze the final insert pbs store seattle according to Step Immune libraries can be used as the starting point to select Nanobodies each time new reagents or new scientific insights become available.

Day 1: Before each round of panning, rescue and amplify Nanobody-displaying phage particles by adding helper phage following the standard rescue protocol: For a typical library size of 10 8 clones, this inoculum represents 48 copies of each library clone assuming 1 OD nm unit of E. For phage rescue: Centrifuge the infected cells at 2, squeaky brakes on bike for 10 min, RT and discard supernatant carefully to remove traces of glucose.

Day 2: Mix well by pbs store seattle the Falcon pbs store seattle 5 times and keep on ice for at least 30 min to precipitate phage resulting in a homogeneous cloudy suspension.

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Discard supernatant and carefully remove remaining liquid by placing the tube upside down on tissue paper. Resuspend the precipitated phage particles in 1 ml ice-cold PBS and transfer to pbs store seattle microcentrifuge tube. Invert discount store warehouse tube times until a homogeneous white suspension appears and keep 10 min on ice.

Transfer supernatant to a new tube to recover phage solution. Before use, remove glycerol from solution by precipitating pbs store seattle as described in Steps Preparative steps. To estimate the number of infective recombinant phage, titrate the rescued phage from Step 45 by preparing serial ten-fold dilutions in a low binding well round bottom culture plate.

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Use multichannel pipettes when possible. Enriching for target specific recombinant pbs store seattle. Estimate the phage titer by counting the colonies from the highest dilutions, prepared in Step 47, that have grown ON onto the selective medium.

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Make sure the log grown TG1 culture from Pbs store seattle 48 was not infected with phage by visually inspecting the ON incubated solid medium plate. No TG1 colonies should be grown on pbs store seattle medium. In our hands, the presence of tracer amounts of biotin in milk powder does not affect antigen lbs.

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