Archives: Methods

(Sandermann and Stromiger. 1972. J. Biol. Chem. 247:5123-5131.)

The advantage of this assay over the Lowry assay from which it is derived, is the presence of SDS in reagent A. This allows one to perform protein assays in nearly any detergent (eg., when detergents are used in the purification of outer membrane proteins). In addition, SDS separates membrane proteins from contaminating membrane constituents and denatures the proteins allowing more reproducible results.

Solutions:

Assay:

1. Wash all glassware with distilled water to remove detergents.
2. In 13mm borosilicate glass test tubes, add 5 and 10ul of a ten-fold dilution of outer membrane preparation.
3. Prepare a standard curve by adding 0, 5, 10, 15, 20 and 25ul of 0.1% BSA in water to test tubes as in step 2. Make sure you prepare 2 samples of 0 BSA so you have enough sample for the reference cuvette (1mg/ml).
4. Add 1ml of solution C, allow to stand for 15 minutes at room temperature.
5. Add 0.1ml of solution D and vortex the test tube immediately, allow to stand for 30 minutes at room temperature.
6. Read at 650nm against a reagent blank.

Procedure

1. Remove an appropriate number of BTSure units from the box.
   • Remove one unit for each area of the autoclave to be tested and one additional unit to be used as a positive control (this one will not be placed in the autoclave).
2. Label indicators with appropriate process information.
3. Place each unit into a small beaker in a horizontal position with representation materials to be sterilized.
   • Cover the beakers with aluminum foil.
4. Place beakers inside the autoclave.
   • One of the beakers should be situated so that it is directly over or next to the drain. The area surrounding the drain is the coolest part of the autoclave and considered to be the least effective area for sterilization.
   • The positive control should not be placed in the autoclave
5. Operate the autoclave per autoclave SOP.
6. Operator shall put the thermal insulated gloves on prior to removing materials from autoclave.
7. Remove the beakers from the autoclave; allow indicators to cool for at least 10 minutes.
8. Remove BTSure biological indicators from the beakers.
   • Observe colour change (blue to black) of the chemical indicator on the BTSure label. NOTE: Colour change indicates exposure to steam. It does not indicate acceptable sterilization.

9. Incubation

   • Place an indicator into the crusher in an upright position and squeeze the crusher to break the glass ampoule. This will allow the strip to be immersed in the media.
   • Repeat the crushing with each additional indicator including the positive control.
   • Immediately place the indicators (including the positive control) into the incubator.
   • Incubate at 55 to 60°C for at least 48 hours.
10. • Examine indicators at regular intervals for any change in colour and record results.
      • The positive control tube should change from purple media to yellow. If it does not, the study is invalid and needs to be repeated with a new lot of indicators.
      • The sample indicators should not change colour. No colour change (media remains purple) indicates adequate sterilization.
      • If the sample indicators turn yellow this indicates bacterial growth and an investigation will need to be conducted. This may include re-performing the autoclave cycle.
    • Report any indication of bacterial growth to instructor or lab manager.
    • Dispose of all used BTSure tubes in a biohazard sharps receptacle when finished.

References:

Christensen, B., Fink, J., Merrifield, R. B., and Mauzerall, D. (1988) Proc. Natl. Acad Sci. U.S.A. 85, 5072-5076

Sims, P. J., Waggoner, A.S., Wang, C.H., and Hoffman, J. F. (1974) Biochemistry 13, 3315-3329

Buffers and Media:

• MH Broth tubes (5ml) (or LB no salt)
• MH Plates (or LB no salt)
• 5mM HEPES, 5mM glucose (E.coli), pH 7.4
• 5mM HEPES, 20mM glucose (S. aureus), pH 7.4
• 400uM DiSC35 stock solution in ethanol
• 4M KCI stock solution

Preparation of Cells:

1. Inoculate 5Ml of LB no salt or MH broth for an overnight culture
2. Use this overnight to prepare a 1 in 100 dilution (50ul in 5ml broth)
3. Grow to an OD600 of 0.5 to 0.6
   1. coli C500, S. aureus C622 = 2-3 hours (check at 1.5 hours)
4. Pellet cells at 3000rpm for 10 min
5. Wash pellet with appropriate buffer
6. Resuspend cells in same buffer to OD600 of 0.05 (usually 40 ml)

E.coli Specific:

1. add DiSC35 to a final concentration of 0.8uM and incubate at room temperature with shaking for 1 hour (1 hour 30 minutes may work better)
2. add KCI to a final concentration of 200mM and incubate at room temperature with shaking for 15 minutes (30 minutes may work better)

S.aureus Specific:

1. add KCI to a final concentration of 200mM or 2mL and incubate at room temperature with shaking for 30 minutes
2. add DiSC35 to a final concentration of 0.8uM or 80uL and incubate at room temperature with shaking for 40 minutes (cover with tin foil)

Assay procedure:

1. settings: Excitation = 622nm, Emission = 670mm, slit width = 10nm, ensure auto cut-off filter is off and stir bar is set to low
2. Add 2ml of cell solution to cuvette and record background fluorescence for 100 seconds
3. Add desired concentration of peptide and continue to measure fluorescence until plateau is reached

•  0.1% Amido Black
• Destaining Solution For Polyacrylamide Gels
• LPS Silver Stain #1
• LPS Silver Stain (Cont.)
• LPS Silver Stain #2
• LPS Silver Stain #2 (Cont.)
• Protein Silver Stain
• Protein Silver Stain (Cont.)

• Basal Medium 2
• Defined Phosphate Minimal Media For E. Coli
• Defined Minimal Media
• Defined Phosphate Minimal Media (Keith’s) – For P. Aeroginosa And Other Bacteria
• Dulbecco’s Minimal Eagle’s Media (DMEM)
• Hybridoma Selective Media
• LB (Luria-Bertani) Medium
• M9 Minimal Medium
• Minigel Recipe For 2 Gels (1 Gel)
• PP2 Medium

ELISA Buffers:

Coating Buffer:

pH 9.6 (Don’t usually need to adjust). Best pH during first 2 weeks.

DO NOT AUTOCLAVE!

Washing Buffer (1 liter):

Note: adding Tween 20 (0.05% vol/vol = 5 ml/1 litre buffer) is optional for some ELISAS but do not use if testing membrane proteins. If using Tween, wash 3X, then wash 1X with no Tween.

Blocking/Conjugate Buffer (1 liter):

Substrate Buffer:

Diethanolamine 100 mls

Conc. HCl (approx. vol.) 10 mls

pH 9.8. Store in the dark, in a dark bottle. The older the buffer, the longer the substrate will take to develop. Make fresh at least every 3 months; more often if necessary.

Store all ELISA buffers at 4^oC.

Remember COATING and SUBSTRATE BUFFERS work best fresh.

ELISA Assay:

Use ELISA 96 well plates. (Currently using Dynatech flexible PVC or Falcon Pro-Bind) Use lids or wrap plates during incubations to avoid evaporation.

Leave 1st vertical row as antigen blank (easiest to blank machine when reading plates). If possible avoid using all “outer wells” of plate.

1. Coat wells with 50 – 100 ul of antigen diluted to 20 – 50 ug/ml in COATING BUFFER.
2. Incubate 1 hr at 37^oC (or overnight at 4^oC).
3. Discard liquid. Wash plates 3X with WASHING BUFFER. Discard all liquid.
4. Add 100 ul of BLOCKING/CONJUGATE BUFFER. Incubate 30 min. at 37^oC.
5. Add 50 ul undiluted culture supernatant or antiserum diluted in BLOCKING/CONJUGATE BUFFER. Incubate 1 hr. at 37^oC.
6. Wash as in Step 2.
7. Add 100 ul of 1/1000 dilution of conjugate in CONJUGATE BUFFER. (Conjugate = Alkaline phosphatase alpha mouse IgG). Incubate 1 hr at 37^oC in dark (alk. phos. = light sensitive)
8. Wash as in Step 2.
9. Add substrate ( 1 Sigma tablet paranitrophenyl phosphate to 5 mls SUBSTRATE BUFFER. Don’t make ahead!). Add 150 ul and incubate at 37^oC in dark.
10. Turn on ELISA plate reader at least 10 minutes before estimated reading time. Read plates after 45 minutes. Time may vary. Read before substrate is intense yellow color (over “1.00” on reader).
11. Read first clones and other weak reactions overnight.

Note:– incubations can be performed 1 hr at 37^oC, 2 or > hr at RT or overnight at 4^oC.

This assay measures the shift in the absorbance maximum of the dye due to binding to endotoxin. The dye is thought to bind to lipid A, causing metachromasia. Sensitivity is approximately 1ug of LPS. Polymixin B (and probably aminogylcosides) interfere with the assay causing quenching. Serum components can also interfere. LPS O-antigen (eg., acid hydrolysed LPS) may not react.

Reference:

Keler and Nowotny. (1986). Analytical Biochemistry. 156:189.

Materials:

• 1.6mg 1,9-dimethylmethylene blue (Serva)
• 0.43g glycine
• 0.33g NaCl
• 4.7ml 1.0M NaOH
• 0.5ml 80% ethanol

Make up to 100ml with distilled water. Stir the above ingredients at room temperature for 3 to 5 hours until the DMB is dissolved. Store the dye solution in a dark bottle at room temperature.

Method:

1. Prepare a standard curve of eg., Shigella LPS. The curve is not linear at high concentrations of LPS.
2. Prepare dilutions of the test LPS at about 10 to 100ug LPS.
3. Add 1ml of dye solution to 50ul of LPS standard or test solution.
4. Mix and read immeadiately at 535nm against a reagent blank (ie., 1ml of dye solution with 50ul of water).

NOTES:

1. Rinse the cuvettes with acetone, then water after every 5 readings to remove the red film.
2. The pH of the dye solution is important (better up than down).
3. Nucleic acids may contribute to the values, but nuclease-treated samples can be tested if this is suspected.

Materials:

Running buffer:

• 2 µl 1.25M Tris-HCl, pH 6.
• 35 µl distilled water
• 2.5 µl 2-mercaptoethanol
• 12.5 µl 10% SDS
• 10 µl 80% glycerol
• 2 µl bromphenol blue

Make up running buffer fresh before use.

TNE buffer:

• 10 mM Tris-HCL, pH 8.0
• 10 mM NaCl
• 0.5 mM EDTA

Methods:

1. Resuspend cell pellet in buffer TNE containing 1% NP40 detergent and vortex.
2. Incubate for 30 minutes on ice or at 37^oC for 10 to 15 minutes.
3. Pellet cell debris in an Eppendorf mini centrifuge (10,000 x g) for 3 minutes.
4. Decant the supernatant into a fresh tube and add 40-50 µl of antiserum.
5. Incubate on ice for 2 hours or overnight at 4^oC.
6. Add 100 µl of S. aureus protein A and 100 µl 5% BSA in TNE.
7. Incubate on ice for 2 hours.
8. Pellet the immune complexes and wash twice with 1ml 1%NP40, 0.5% Na deoxychloate, 0.1% SDS in TNE and sonicate once to resuspend.
9. Resuspend the final pellet in 70 µl running buffer.
10. Boil in a water bath for 90 seconds and centrifuge for 1 minute to remove S. aureus (saving supernatant).
11. Refrigerate, if the preparation is to be stored before use (e.g. SDS-PAGE).

1. Kill immunized mouse by CO₂ asphyxia and wet throughly with ethanol. Aseptically remove spleen and place on sterile cell sieve (screen).
2. Cut the spleen into small pieces with sterile scissors and press through the screen with the end of the plunger of a 1ml sterile syringe into a sterile petri dish containing 10ml of DMEM-10%FCS. This is done gently as pressing too forcefully increases the amount of connective tissue present, increasing the chance that the hybridomas will be overgrown by fibroblasts.
3. Aspriate the cells once up and down a 10ml syringe with a 22g needle.
4. Transfer the cells to a sterile 50ml Falcon tube and allow to settle for 5 minutes.
5. Resuspend NS-1 cells from the tissue culture flasks and decant into 50ml sterile Falcon tubes.
6. Remove the supernatant from the spleen cell suspension, leaving the large clumps and put into a fresh Falcon tube.
7. Pellet the spleen cells and NS-1 cells for 5 minutes at 1200rpm.
8. Remove the supernatants and resuspend the cells separately in 10ml of DMEM-10%FCS.
9. Dilute each cell suspension 1:10 in 1% trypan blue in PBS. Count using the haemocytometer.
10. Pellet the cells for 5 minutes at 1200rpm and combine the spleen and NS-1 cells at a ratio of 10:1 (ie., 10⁸ spleen cells to 10⁷ NS-1 cells).
11. Mix gently and and spin at 1200rpm for 7 minutes.
12. Remove supernatant and add 0.5ml 41.6% PEG₁₅₅₀ to the cells with gentle stirrring over exactly 1 minute. Rock the tube for 2-3 minutes, add 0.5ml 25% PEG₁₅₅₀ over exactly 1 minute and rock the tube for a further 2-3 minutes.
13. Add 4ml of DMEM slowly and add a further 20ml of DMEM-20%FCS gently and incubate at 37^oC for 2-3 hours.
14. Prepare thymus feeder cells. Aseptically remove the thymus from a young mouse, prepare a cell suspension as for spleen cells and place in 50ml of DMEM-20%FCS. The thymus of old or large mice should be prepared in 20ml DMEM-20%FCS.
15. Mix the spleen-NS-1 cells with the thymocytes and transfer 1.0ml aliquots to 24 well NUNC tissue culture trays, or 0.1ml aliqouts to 96 well NUNC tissue culture trays.
16. Check after 24 hours for contamination and discard contaminated plates.
17. Prepare selective HAT medium (ie., DMEM-20%FCS with HAT). Remove most of the medium in the wells without disturbing the cells and replace with 1.0ml of HAT medium.
18. After 4 days replace the medium in the wells with fresh HAT medium without disturbing the cells.
19. Wait and screen visually for hybridomas.

Hybridomas can be tested by ELISA when they cover approximately one third of the well. Do not allow the cells to overgrow as antibody secreting cells will die off first.

Reference:

Ouchterlony, (1959).

Materials:

0.08% barbitone buffer:

1. 12.0 g sodium 5’5 diethyl barbiturate
2. 4.40 g 5’5 diethylbarbituric acid
3. Adjust the pH to 8.2 with 1.0 M NaOH and make up to 1 litre with distilled water.

Methods:

1. Prepare a 2% (w/v) agarose (Standard low Mr agarose, Biorad) suspension in 0.08% barbitone buffer.
2. Add Triton X-100 to a concentration of 1% (v/v) and pour 8ml of the agarose suspension onto a glass slides.
3. The outer antigen wells are filled with 20ug of the solubilized antigen and the centre antiserum well is filled with undiluted or two-fold dilutions of antiserum.
4. The agarose slides are incubated for 16 hours at 37^oC in a humid chamber.
5. The wells are filled with PBS, and the slides are incubated at 4^oC for a further 72 hours.
6. The slides are pressed dry under a stack of Whatman 3M filter papers, washed five times in PBS for 45 minutes at 37^oC, dried again, stained in 1% (w/v) Coomassie brilliant blue dissolved in absolute methanol, acetic acid and water at a ratio of 4:1:4 and destained in the same solution without the dye.