구체적인 단백질 실험 프로토콜

NO1. Antibody purification on protein A or protein G columns

Antibody source	Protein A	Protein G
Monoclonal antibodies    mouse IgG1    Mouse IgG2a,IgG2b,IgG3    rat	V	V  V
Polyclonal antibody     human     rabbit     mouse    rat    horse    goat    donkey    pig    guinea pig    dog    cow	V  V    V    V  V  V  V  V	V  V  V

1.  Adjust the pH of the crude antibody preparation by adding 1/10 volume of 1.0 M Tris(pH8.0)

2.  Pass the antibody solution through a protein A or protein G bead column. These columns bind approximately 10-20 mg of antibody per milliliter of wet beads. Serum contains approximately 10mg/ml of total IgG, tissue culture supernatant contain 20-50 μg/ml of monoclonal antibody, and ascites between 1 and 10mg/ml.

     Note the approximate volume of the column bed because the wash and elution buffer are measured in relative values compared to the volume of beads being used.

3.  Wash the beads with 10 column volumes of 100mM Tris(pH8.0)

4.  Wash the beads with 10 column volumes of 100mM Tris(pH8.0)

5.  Elute the column with 50 mM glycine (pH3.0). Add this buffer stepwise as approximately 1/2 column volume per step. Collect the elute from each step in tubes containing 1/10 column volume of 1M Tris (pH8.0). Mix each tube gently to bring the pH back to neutral.

6.  Identify the immunnoglobulin-containing fractions by absorbance at 280nm. (1OD=approximately 0.8mg/ml)

 Combine the antibody-containing fractions and measure purity by running 2μg of total protein on a SDS-polyacrylamide gel and staining with Coomassie blue. Good purification should yield essentially pure heavy and light chain bands.

No2. Staining cells growing on coverslips 

Dection limits is approximately 1,000-10,000 molecules/cell if the antigen is localized.

Summary
 
 Rapid and easy
 Detects antigen presence and localization
 Qualitive to semi-quantitative
 Sensitivity dependent on minimal level and localization of antigen
 High local concentration of antigen, so lower affinity antibody OK

Caution
 Ethanol, FITC, fluroscein, paraformaldehyde

1. Sterilize glass coverslips prior to cell staining. Place round coverslips (grade #1 or #1.5) in 70% ethanol.
    Remove individually and flame to sterilize.

2. Aseptically transfer the coverslips into a sterile tissue-culture dish by using a sterile pasteur pipet connected to a vacumn line. A light vacumn flow through the pipet will allow the coverslips to be picked up and moved easily.

3. Plate the cell suspension in the tissue-culture dish at low density. Grow overnight.

4. Remove the cell suspension in the tissue-culture media by aspiration and wash the coverslips once with PBS. Sterility is not needed at this or later steps.

5. Asprate the wash buffer and fix the cells by adding 4% paraformaldehyde. Incubate for 10 minutes at room temperature.

6. Remove the paraformaldehyde by aspiration and wash the cells twice with PBS.

7. Aspirate the last wash buffer and permeabilize the cells by adding 0.2% Triton X-100 in PBS. Incubate for 5 minutes at room temperature.

8. Remove the detergent solution by aspiration. Wash the coverslips in 0.2% Triton X-100 in PBS with three changes over 5 minutes. Drain well but do not allow the specimens to dry. Move each coverslips to the well of a 24-well tissue-culture plate.

9. Add the primary antibody solution to the coverslips. Use 25 ul to cover the entire coverslip. Make sure the coverslips does not touch the side of the well.

   Monoclonal antibodies are most often used as undiluted tissue-culture supernatants. Ascites fluids, purified monoclonal and polyclonal antibodies, and crude polyclonal sera need to be checked for the proper dilution prior to use. To determine the proper titration of the antibody to use, test 1/10, 1/100, 1/1000, and 1/10,000 dilutions of the starting antibody solution. All dilutions should be done in 0.2% Triton X-100 in PBS containing 3% BSA.

   Each assay should include three controls: (1) An irrelevant antibody from the same species and type as the primary antibody to determine the specificity of the staining. (2) A sample with no primary antibody to test for the background of the labeled secondary antibody. (3) If possible, a known control against a positive.

   Incubatefor 60 minutes at room temperature.

10. Wash the coverslips in three changes of 0.2% Triton X-100 in PBS over 5 minutes. After the last wash, drain well but do not allow the specimens to dry.

11. Apply the labeled secondary reagent. Add 25ul.

     A useful secondary reagent is anti-immunoglobulin antibodies conjugated to FITC. All labeled secondary reagents need to be checked for the proper dilution prior to use. To determine the proper titration of these reagents to use, test 1/10, 1/100, 1/1000, and 1.10,000 dilutions of the starting material. All dilutions should be done in 0.2% Triton X-100 in PBS containing 3% BSA.

     Incubate for 20 minutes at room temperature.

12. Wash the coverslips in three changes of 0.2% Triton X-100 in PBS over 5 minutes. Drain well.

13. On a clean and labeled microscope slide place a drop (approximately 50 ul) of mounting medium.
     Remove the coverslips from the dish using fine-tipped forceps and drain the last of the wash buffer by touching the edge of the coverslip to a clean paper towel. Invert the coverslip and place on the dropr of mounting medium with the cell side down. Gently lower the coverslip, touching one edge to the slide next to the drop. Then allow the coverslip to fall on the drop. This will push any bubbles ahead of the falling coverslip. Allow to air dry for at least 30 minutes prior to observing.

14. Observe and photograph under the fluorescence microscope.



No 3. Frozen tissue sections.

Detection limit is approximately 1,000-10,000 molecules/cell if localized.

    Detects antigen presence and localization
    Qualititive to semi-quantitative
    Sensitivity dependent on minimal level and localuzation of antigen
    High local concentration of antigen, so lower-affinity antibody

Cautions

    Diaminobenzidine (DAB), DPX, ethanol, hydrogen peroxide, nickel chloride, pataformaldehyde, sodium azide

1. Dissect a small sample. no bigger than approximately 1 cm2 * 0.4 cm, of undamaged tissue.

2. Place the specimen on one end of a strip of card. Label the card, and submerge the end with the sample in liquid nitrogen. After 60 seconds in the liquid nitrogen, remove the sample and place on dry ice. Trim the card to just larger than size of the tissue and transfer to a precooled (-70°C), labeled freezing vial.

3. Prior to sectioning, coat clean glass slides with 1% gelatin. Dissolve gelatin in water by heating to 50°C, cool, and add sodium azide to 0.02%. Dip the slides in the solution for 30 seconds, remove, and allow to air-dry.

4. Prepare sections of frozen tissues using a cryostat followinf the manufacturer's direcions. Sections between 5 um and 10 um commonly are used. Collect the sections on the coated slides.

5. Allow the sections to air-dry. Dip in freshly prepared 4% paraformaldehyde for 2 minutes.

6. Wash with several changes of PBS, and place in 1% NP-40, PBS, for 5 minutes. Wash with several changes of PBS.

7. Place slides with the tissue sections in a humidified chamber. Add the first antibody solution. All dilutions must be carried out in protein-containing solutions such as 3% BSA in PBS.

    Monoclonalantibodies are best applied as tissue-culture supernatants (specific antibody concentration of 20-50 ug/ml, use neat). Ascites fluids, purified monoclonal and polyclonal antibodies, and crude concentrations between 0.1 and 10 ug/ml. If the specific antibody concentration of the antibody sample is unknownm prepare and test 1/10, 1/100, 1/1000, 1/10000 dilutions of the starting material.

8. Incubate the slides for a minimum of 60 minutes at room temperature in the humidified chamber. For some reactions, prolonged incubations of up to 24 hours can increase sensitivity.

9. Wash in three changes of PBS over 5 minutes.

10. Apply the horeseradish perixidase-labeled secondary reagent specific for your promary antibody. These reagents can be purchased from several suppliers.
     If the correct amount of secondary reagent is not known, test dilutions of the secondary antibodies 1/50 to 1/1000. Carry out all dilutions in a protein-containing solution such as 3% BSA/PBS.

11. Incubate with the labeled secondary reagent for 30 minutes at room temperatur in the humidified chamber.

12. Wash in three changes of PBS over 5 minutes.

13. Prepare the DAB/metal reagent. Dissolve 6 mg of DAB in 9ml of 0.05M Tris buffer (pH 7.6). Add 1 ml of a 0.3% wt/vol stock solution of nickel chloride inwater (the same amount of cobalt chloride can be used as an alternative). Add 0.1 ml of a 3% solution of hydrogen peroxide i water. If a precipitate appears, dilter through Whatman No. 1 filter paper (or equivalent).

14. Apply the solution to the specimen. Observe the sample inder a low-power light microscope. When the bown/ black precipatate has developed sufficiently, stop the reaction by washing in water. This will normally be between 1 and 20 minutes.

15. Add a few drops of Harris' Hematoxylin to the specimen. Incubate for approximately 5 minutes. The length of time will determine the intesnsity of the stain.

16. Wash the slide gently in water.

17. Dehtdrate the sample by passing through graded alcohols. Incubate twince for 3 minutes each in 75% ethanol, twice for 3 minutes each in 95% ethanol, and twice for 3 minutes each in absolute ethanol. Air-dry.

18. Add a small drop of DPX to the specimen. Carefully place a coverslip (#1) on the drop, avoiding air bubbles. Remove any excess mounting medium with a paper towel.
     DPX will set almost immediately and the samples can be observed and photographed.



No4. Immunoprecipitation

Summary
     Determines antigen presence, quantity, size, turnover, modifications, associations
     Multiple steps over 1/2 to 1 day
     Semi-quantitative to quantitative
     Sensitivity dependent on relative quantity of antigen and affinity of antibody
     Needs high affinity/ avidity antibody

1. Add 50 ul of normal rabbit serum per 1.0 ml of antigen solution.

2. Incubate 1 hour on ice.

3. During this incubation, wash fixed S.aureus Cowan (SAC) once in lysis buffer. Use 100 ul of packed SAC per 50ul of normal rabbit serum. It is easiest to spin SAC briefly at 10,000g for 30 seconds and resuspend the pelleted SAC by triturating with a pasteur pipet. Spin again at 10,000g and remove the wash buffer. Set the packed pellet of washed SAC aside on ice.

4. After the 1-hour incubation, use the lysaste to resuspend the washed pellet of SAC.

5. Incubate 30 minutes on ice.

6. Spin at 10,000g for 15 minutes at 4°C. Carefully remove and save the supernatant.

7. Divide the supernatant and aliquot into the appropriate number of 1.5 ml conical tubes. To invidual tubes, add the immune or control serum (1ul), hybridoma tissue-culture supernatant (50ul), or ascites fluid (1ul). Over volumes will be appropriate in specific settings as determined by titration of antibody volume versus a constant antigen amount.

8. Incubate on ice for 1 hour.

9. To the antibody-antigen reaction, add protein A or protein G beads depending on the type of antibody used. Add 100 ml of a 10% vol/vol slurry made in lysis buffer (final bead volume will be 10 ml). Close the caps securely. Incubate 1 hour at 4°C will rocking.

10. Collect the beads by centrifugation at 10,000g for 15 seconds at 4°C. Wash the immune complexes three times with lysis buffer. The lysate and wash buffers are easily removed by aspiration through a 23-gauge needle.

11. Remove the final wash as completely as possible.

12. Use the immune complexes for the appropriate assay, often SDS-polyacrylamide elextrophoresis.

Antibody source	Protein A	Protein G
Monoclonal antibodies    mouse IgG1    Mouse IgG2a,IgG2b,IgG3    rat	V	V  V
Polyclonal antibody     human     rabbit     mouse    rat    horse    goat    donkey    pig    guinea pig    dog    cow	V  V    V    V  V  V  V  V	V  V  V

No5. Immunoblotting

Summary 

     Detection limit is 2-20 fmoles, or about 0.1-1 ng of a 50-kD protein
     Multiple steps over 2 days (or 1 long day)
     Detects antigen presence, quantity, size
     Semi-quantitative to quantitative
     Antigen detection dependent on denaturation-resistant epitope
     In general, lower-affinity antibody may work

Caution

DTT, methanol, SDS

1. Solubilize your proteins in Lasmmli sample buffer. Final sample buffer conditions should be 2% SDS, 100 mM dithiothreitol (added fresh from a 1M stock solution, which is kept frozen at -20°C), 60 mM Tris (pH 6.8), 0.01% bromophenol blue, and 10% glycerol. Whole cells can be solubilized directly in sample buffer; purified or partially purified solutions can be diluted 1:1 with 2% sample buffer. Heat to 70°C for 10 minutes. The final protein concentration should not exceed 150 ug/well (15 ug/well for minigels).

2. Run your samples on a SDS polyacrylmide gel, remove the plates, and cut the gel to the desired size for transfer. Mark the gel to estabilish orientation.

3. Cut one sheet of nitrocellulose paper and four sheets of absorbent filter paper (Whatman 3MM or equivalent) to the size of the gel.

4. Wet the nitrocellulose membrane in distilled water. Move the membrane to soak in transfer buffer for 5 minutes. Wet the absorbent paper by soaking in transfer buffer. Transfer buffer 1 (proteins 20,000-400,000 kD) : 48 mM Tris, 390 mM glycine, 0.1% (wt/vol) SDS, 20% methanol, and distilled water to 1000 ml. Transfer buffer 2 (proteins < 80,000 kD) : 25 mM Tris, 190 mM glycine, 20% methanol, and distilled water ro 1000 ml.

5. Immerse gel, membranes, folter papers, and support pads in transfer to ensure that they are throughly soaked. Be careful to exclude air bubbles from the support pads.

6. Assemble the transfer sandwich : Support pad, 2 sheets absorbent paper, gel, filter, 2 sheets absorbent paper, support pad. Place the complete sandwich in the transfer tank with membrane closet to the positive electrode (anode, red electrode)

7. Transfer overnight at 4°C. For proteins over 100,000 kD, transfer at 28 colts for 1 hour, then at 84 volts for 14-16 hours. For proteins under 100,000 MW, transfer at 63 volts for 4-16 hours.

8. After transfer, disconnect the power supply. Disassemble the sandwich and mark the nitrocellulose membrane to retain orientation.

9. Rinse the folter several times with PBS.

10. Incubate the blot in 5% nonfat dry milk / PBS at room temperature with agitation for 2 hours. Then remove the blot from the blocking solution and wash twice for 5 minutes each in PBS.

11. Add the primary antibody solution. Use 10 ml per 15 * 15 cm blot. All dilutions should be done in 3% BSA/PBS.

12. Incubate the blot with antibody for 1 hour at room temperature with agitation. Wash the blot with four changes of PBS for 5 minutes each.

13. Add horseradish peroxidase-labeled secondary reagent. All dilutions should be done in 3% BSA/PBS. Use tissue-culture supernatants either undiluted or diluted up to 1 in 10. This yields an antibody concentration between 1 and 50 ug/ml. For polyclonal sera or ascites fluid, a dilution of 10 ul in 10 ml will normally be sufficient. If the concentration of an antibody solution is unknown, try several dilutions to determine the correct range.

14. Incubate for 1 hour at room temperature with agitation. Then wash the blot with foir changes of PBS for 5 minutes each.

15. Immediately prior to use, prepare fresh chemiluminescence reagent. Prepare only as much reagent as needed, because the reagent has a very short chelf life. Incubate with the blot for 1 minute.

16. Drain the blot and remove excess chemiluminescence reagent by blotting the edge or corner of the filter with a paper towel. Place the blot in plastic wrap to ensure a dry surface for film exposure.

17. Expose the blot to film in the dark room for 1 minute. Develop the film and determine the correct exposure time for your antigen. Exposure times may range from a few seconds to several hours.



No6. Immunoaffinity purification

Summary

Purification factor is 1,000-to 10,000-fold
Rapid antigen purification, columns often reusable
Yields purified antigen
Not useful for quantitation
Efficiently depends on antigen concentration and antibody affinity
Needs moderate affinity antibodyeral, lower-affinity antibody may work

Caution
DMP, ethanolamine, SDS

1. Bine the antibody tp protein A or protein G beads. For general-purpose columns, bind approximately 2 mg of monoclonal antibody or affinity-purified polyclonal antibodies per milliliter of wet beads. Mix the antibodies and protein A or G beads together in a loose slurry, using about 1 ml of bead volume per 10 ml of total solution. Incubate at room temperature for 1 hour with gentle rocking.

2. Wash the beads twice with 10 volumes of 0.2M sodium borate (pH 9.0) by centrifugation at 3000g for 2 minutes or 10,000g for 30 seconds.

3. Resuspend the beads in 10 volumes of 0.2 M sodium borate (pH 9.0) and remove and place aside the equivalent of 10 ul of wet bead volume. Add enough dimethyl pimelimidate (solid) to the total bead slurr to bring the final concentration to 20 mM.

4. Incubate for 30 minutes at room temperature with gentle mixing. Remove the equivalent of 10 ul of the coupled beads.

5. Stop the reaction by washing the beads once in 0.2M ethanolamine (pH 8.0) and then incubate for 2 hours at room temperature in 0.2M ethanolamine with gentle mixing. The beads can be stored at this stage by washing in PBS and sorting in 0.01% Merthiolate in PBS.

6. Checj the efficiently of coupling by boiling samples of beads taken before and after coupling in Laemmli sample buffer. Run the equivalent of 1 ul and 9 ul of both samples on a 10% SDS-polyacrylamide gel and stain with Coomassie blue. Good coupling is indicated by heavy-chain bands (55,000 MW) in the "before" but not in the "after" lanes.

7. Transfer the antibody beads to a suitable column. Rinse the mixing chamber with PBS to collect the remaining beads. If possible, use only as much antibody-bead matrix as needed to bind the total amount of antigen in the preparation.

8. Wash the column with 20 bed-volumes of buffer used to prepare the antigen.

9. Apply the antigen solution to the column. Pass the solution through the column with a flow rate of approximately 1 ml/hour per each 1 ml of column volume. This is easiest to control using a pump.

10. Wash the column with 20 bed-volumes of binding buffer.

11. Change the buffer in the column to the pre-elution buffer by washing with 20 column bed-volumes. 

12. Using a stepwise elution, sequentially pass 0.5 bed-volume of the elution buffer through the column. Collect each fraction in seperate tubes. Ig either high or low pH is used to elute the column, the collection tubes should contain 0.1 bed-volume of neutralizing buffer.

13. Check each tube for the presence of the antigen. Combine tubes with high concentrations. Depending on how the antigen will be used, it may be necessary to dialyze the resulting eluted proteins to change the buffer.

14. Return the column to the starting buffer by passing 20 column volumes through the matrix. Add 0.01% Methiolate for long-term storage(4°C). 



No7. Preparation of stock solution

Molecular  Weight (daltons)	1 ug	1 nmole
100	10 nmoles or   6*10의 15제곱 molecules	0.1 ug
1,000	1 nmole or  6*10의 14제곱 molecules	1 ug
10,000	100 pmoles or  6*10의 13제곱 molecules	10 ug
20,000	50 pmoles or  3*10의 13제곱 molecules	20 ug
30,000	33 pmoles or  2*10의 13제곱 molecules	30 ug
40,000	25 pmole or   1.5*10의 13제곱 molecules	40 ug
50,000	20 pmoles or  1.2*10의 13제곱 molecules	50 ug
60,000	17 pmoles or  10의 13제곱 molecules	60 ug
70,000	14 pmoles or   8.6*10의 12제곱 molecules	70 ug
80,000	12 pomoles or  7.5*10의 12제곱 molecules	80 ug
90,000	11 pmoles or  6.6*10의 12제곱 molecules	90 ug
100,000	10 pmoles or  6*10의 12제곱 molecules	100 ug
120,000	8.3 pmoles or  5*10의 12제곱 molecules	120 ug
140,000	7.1 pmoles or  4.3*10의 12제곱 molecules	140 ug
160,000	6.3 pmoles or  3.8*10의 12제곱 molecules	160 ug
180,000	5.6 pmoles or  3.3*10의 12제곱 molecules	180 ug
200,000	5 pmoles or  3*10의 12제곱 molecules	200 ug

David's life chart 3

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 Some useful nucleotide dimensions

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 1cm of DNA ≈3 * 10의 6제곱 nucleotides

Organism	Base pairs/  haploid genome	Base pairs/  diploid genome	Length / cell	Mass
Human	3 * 10의 9제곱	6 * 10의 9제곱	2 meters (diploid)	6 pg
Fly	1.65 * 10의 8제곱	3.3 * 10의 8제곱	100 cm (diploid)	0.3 pg
Yeast	1.35 * 10의 7제곱	2.7 * 10의 7제곱	10 cm (diploid)	0.03 pg
E.coli	4.7 * 10의 6제곱	-	1.5 cm (haploid)	0.0045 pg
SV40	5 * 10의 3제곱	-	1.7 nm	0.000006 pg

Some useful cell dimensions

Organism	Dimensions	Volume
S.cerevisiae	5 um	66 um³
S. pombe	2 * 7 um	22 um³
Mammalian cell	10-20 um	500-4,000 um³
E.coli	1 * 3 um	2 um³
Mammalian mitochondrion	1 um	0.5 um³
Mammalian nucleus	5-10 um	66-500 um³
Plant chloroplast	1 * 4 um	3 um³
Bacteriophage lambda	50 nm (head only)	6.6 * 10의 -5제곱 um³
Ribosome	30 nm diameter	1.4 * 10의 -5제곱 um³
Globular monomeric protein	5 nm diameter	6.6 * 10의 -8제곱 um³

Some useful concentrarions

Total cell protein concentrations	Detergent soluble protein = 1-2 mg/10의 7제곱 mammalian  cells or 100 -200 mg/ml for soluble proteins only
Specific protein concentrations
Nucleus (200 um³):  Abundant transcription factor  Rare transcription factor	1nM (100,000 copies / nucleus)  10 pM (1,000 copies / nucleus)
Serum	50 - 100 mg / ml

Solution	Method of preparation	Comments
Phosphate-buffered saline (PBS)	Dissolve 8.0g NaCl, 0.2g of KCl, 1.44g of  Na2HPO4, and 0.24g of KH2PO4 in 800ml of distilled H2O.   Adjust the pH to 7.4.   Adjust  the volume to 1L.   Dispense in convenient volumes and sterilize by autoclaving.   Store at room temperature.	Can be made as a 10X stock.
Tris-buffered saline (TBS) (25mM Tris)	Dissolve 8.0g of NaCl, 0.2g of KCl, and 3g of  Tris base in 800ml of distilled H2O.   Adjust the pH to 8.0 with 1M HCl.   Adjust the volume to 1 liter.  Dispense in convenient volumes and sterilize by  autoclaving.   Store  at  room temperature.
10% Sodium azide	Dissolve 10g of sodium azide in 100ml of distilled H2O.   Store  at  room temperature.	Do not use sodium azide for  experiments  using live organisms or for reactions that  use  horseradish  peroxidase.
3% Bovine serum albumin  in phosphate-buffered  saline (3% BSA/PBS)	Add 3g of BSA (Fraction V) to 100ml of PBS.  Allow to dissolve.   Add o.2 ml of 10% sodium azide.  Store at 4°C.
1M Tris	Dissolve 121g of Tris base in 800ml of distilled H2O.  Adjust to the desired pH by adding concentrated HCl.  Adjust the volume to 1000ml with distilled H2O.  Dispense in convenient volumes and sterilize by autoclaving.   Store  at  room temperature.
500mM EDTA	Add 186g of disodium ethylene diamine tetraacetate-2H2O  to 400ml of distilled H2O.   Add NaOH to adjust the pH to 8.0 and to allow the EDTA to dissolve.   Bring volume to   500ml with  distilled H2O.   Dispense in convenient volumes and sterilize by autoclaving.  Store  at  room temperature.
100% (wt/volume)     Trichloroacetic   acetic acid (TCA)	Add 227ml of distilled H2O to a 500-g bottle of TCA
30% Acrylamide mix	Dissolve 29.2g of acrylamide (electrophoresis frade)  and 0.8g of N, N'-methylene-bisacrylamide (electrophoresis grade) in 80ml of distilled H2O.    Adjust the volume to 100ml.    Store at room temperature.
1.5M Tris (pH 8.8)	Dissolve 181.5g of Tris base in 800ml of distilled H2O.  Adjust the pH to 8.8 with concentrated HCl.  Adjust the volume to 1 liter.  Dispense in convenient volumes and sterilize by autoclaving.  Store at room temperature.
1.0M Tris (pH 6.8)	Dissolve 12.1g of Tris base in 80ml of distilled H2O.  Adjust the pH to 6.8 with concentrated HCl.  Adjust the volume to 100 ml.  Dispense in convenient volumes and sterilize by autoclaving.  Store at room temperature.
10% Sodium dodecyl sulfate (SDS)	Dissolve 10g of SDS in 100ml of distilled H2O.  Store at room temperature.
10% Ammonium persulfate (APS)	Dissolve 0.5g of ammonium persulfate (electrophoresis grade) in 5ml of distilled H2O.   Store at 4°C.	Make fresh solution weekly.
2X Laemmli sample buffer	Add 4ml of 10% SDS, 2ml of glycerol, and 1.2ml of 1M Tris (pH6.8) to 2.8ml of distilled H2O.  Add 0.01% bromophenol blue as a tracking dye.  Store at room temperature.	To prepare 1X sampole buffer, mix 5 parts 2X, 4 parts water, and 1 part 1M DTT.
1M Dithiothreitol	Dissolce 5g of DTT in 32ml of distilled H2O.  Dispense in 1-ml aliquots.   Store at -20°C.
10X Laemmli running buffer	To a 10;liter carboy, add 8 liters of distilled H2O, 303g of Tris base, 1442g of glycine, and 100g of SDS.  After all the chemicals have dissolved, adjust the pH to 8.3.   Adjust tje vollume ro 10 liters with H2O.  Store at room temperature.
Destain	To a 10-liter carboy add 2.5 liters of methanol and 700ml of glacial acetic acid.  Adjust the colume to 10 liters with H2O.  Store at room temperature.
4% Paraformaldehyde	Dissolve EM grade paraformaldehyde in PBS in Pyrex container with stir bar (4g to 100ml)  Add a few drops of NaOH and heat in a hood (keep bottle cap loose_ at 60 °C to dissolve.  Cool to room temperature and adjust pH to 7.4.  Make fresh prior to use.
Gelvatol	Dissolve 0.35g of Gelvatol in 3ml of deionized H2O and 1.5ml of glycerol. (PBS can be substituted for water)  Heat the solution with stirring in a boiling water bath until the gelvatol is completely dissolved.  Add anti-fade agents as desired.   Store at 4 °C  (can be stored for months).
Glycerol anti-fade mounting medium	Dissolve in 100ml og glycerol: 5g of n-propyl gallate, 0.25g of DABCO, 2.5mg of p-phenylenediamine.  Add several pellets of NaOH to bring pH above neutral.  Stir thoroughly (>1day).  Store aliquots at -20°C wrapped in foil.
1M NaCL/ 0.05M sodium phosphate (pH7.5)	For liter: Combine 200ml of 5M NaCl, 500 ml of 0.1M sodium phosphate(pH 7.5), and 300ml of H2O.
NP-40 lysis buffer	For 1 liter: Combine 30 ml of 5M NaCl, 100ml of 10% NP-40, 50ml of 1M Tris (pH 8.0), and 820 ml of H2O.  Store at 4°C.
RIPA buffer	For 1 liter: Combine 30 ml of 5M NaCl, 100ml of 10% NP-40, 50ml of DOC, 100ml of 10% SDS, 50ml of 1M Tris (pH 8.0), and 670ml of H2O.  Store at 4°C.

No8. Solutions for Tris/ glycine SDS-polyacrylamide gel electrophoresis

6%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O  30% Acrylamide mix  1.5M Tris (pH 8.8)  10% SDS  10% APS  TEMED	2.6  1.0  1.3  0.05  0.05  0.004	5.3  2.0  2.5  0.1  0.1  0.008	7.9  3.0  3.8  0.15  0.15  0.012	10.6  4.0  5.0  0.2  0.2  0.016	13.2  5.0  6.3  0.25  0.25  0.02	15.9  6.0  7.5  0.3  0.3  0.024	21.2  8.0  10.0  0.4  0.4  0.032	26.5  10.0  12.5  0.5  0.5  0.04
8%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O  30% Acrylamide mix  1.5M Tris (pH 8.8)  10% SDS  10% APS  TEMED	2,3  1.3  1.3  0.05  0.05  0.003	4.6  2.7  2.5  0.1  0.1  0.006	6.9  4.0  3.8  0.15  0.15  0.009	9.3  5.3  5.0  0.2  0.2  0.012	11.6  6.7  6.3  0.25  0.25  0.015	13.9  8.0  7.5  0.3  0.3  0.018	18.5  10.7  10.0  0.4  0.4  0.024	23.2  13.3  12.5  0.5  0.5  0.03
10%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O  30% Acrylamide mix  1.5M Tris (pH 8.8)  10% SDS  10% APS  TEMED	2.0  1.7  1.3  0.05  0.05  0.002	4.0  3.3  2.5  0.1  0.1  0.006	5.9  5  3.8  0.15  0.15  0.006	7.9  6.7  5.0  0.2  0.2  0.008	9.9  8.3  6.3  0.25  0.25  0.01	11.9  10.0  7.5  0.3  0.3  0.012	15.8  13.3  10.0  0.4  0.4  0.016	19.8  16.7  12.5  0.5  0.5  0.02
12%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O  30% Acrylamide mix  1.5M Tris (pH 8.8)  10% SDS  10% APS  TEMED	1.6  2.0  1.3  0.05  0.05  0.002	3.3  4.0  2.5  0.1  0.1  0.004	4.9  6.0  3.8  0.15  0.15  0.006	6.6  8.0  5.0  0.2  0.2  0.008	8.2  10.0  6.3  0.25  0.25  0.01	9.9  12.0  7.5  0.3  0.3  0.012	13.2  16.0  10.0  0.4  0.4  0.016	16.5  20.0  12.5  0.5  0.5  0.02
15%	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O  30% Acrylamide mix  1.5M Tris (pH 8.8)  10% SDS  10% APS  TEMED	1.1  2.5  1.3  0.05  0.05  0.002	2.3  5.0  2.5  0.1  0.1  0.004	3.4  7.5  3.8  0.15  0.15  0.006	4.6  10.0  5.0   0.2  0.2  0.008	5.7  12.5  6.3  0.25  0.25  0.01	6.9  15.0  7.5  0.3  0.3  0.012	9.2  20.0  10.0  0.4  0.4  0.016	11.4  25.0  12.5  0.5  0.5  0.02
STACK	1ml	2ml	3ml	4ml	5ml	6ml	8ml	10ml
H2O  30% Acrylamide mix  1.0M Tris (pH 6.8)  10% SDS  10% APS  TEMED	0.68  0.17  0.13  0.01  0.01  0.001	1.4  0.33  0.25  0.02  0.02  0.002	2.1  0.5  0.38  0.03  0.03  0.003	2.7  0.67  0.5  0.04  0.04  0.004	3.4  0.83  0.63  0.05  0.05  0.005	4.1  1.0  0.75  0.06  0.06  0.006	5.5  1.3  1.0  0.08  0.08  0.008	6.8  1.7  1.25  0.1  0.1  0.01