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U.S.A.实验室WESTELacrosseN,聚芳烃酰胺凝胶电泳

文章作者:生命科学 上传时间:2019-08-27

核心提示:A gel electrophoresis system used for SDS-PAGE protein analysis. The gels are made up of Bis-Tris-HCl polyacry

核心提示:WESTERN BLOT PROTOCOLIn a Western blot, proteins that are separated on polyacrylamide gels on the basis of size are t

核心提示:This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be awar

A gel electrophoresis system used for SDS-PAGE protein analysis. The gels are made up of Bis-Tris-HCl polyacrylamide and are intended for denaturing conditions only.

WESTERN BLOT PROTOCOL

This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

NuPAGE Electrophoresis Protocols

In a Western blot, proteins that are separated on polyacrylamide gels on the basis of size are transferred to a membrane for detection with antibodies.

Solutions

1) Remove gel from pouch and rinse gel with D.I. water holding the gel by the edges of the cassette.

Preparation of Human Cell Extracts

TIP: Use electrophoresis grade reagents to prepare the following solutions:

A: 50 ml IEF Lysis Buffer

  1. Add 21 g urea to 35 ml HPLC-grade H2O to a 50 ml Falcon tube .
  2. Vortex vigorously for several minutes.
  3. Add:

    7.6 g

    Thiourea

    2 g

    Chaps

    0.5 g

    Mega 10

    0.5 g

    OBG

    250 µl

    Triton X-100

    0.25 g

    Tris

    0.4 g

    DTT

    500 µl

    Pharmalytes or IPG buffer pH 3-10

    500 µl

    b-mercaptoethanol
  4. Add 10 µl tributylphosphine 2 mM, under the hood

  5. Add Bromophenol Blue as indicator.
  6. Check volume is 50 ml.
  7. Vortex until all is dissolved .
  8. Aliquot 1 ml into microfuge tubes.
  9. Store at -20°C.

B: 10X Electrophoresis Running Buffer 0.25 M Tris, 1.92 M glycine, 1M SDS

  1. Add 300 g Tris-base, 1441 g glycine, and 10 g SDS to ~7 L HPLC-grade H2O.
  2. Mix gently until dissolved.
  3. Bring volume to 10 liters.

2) Mark the bottom of the lanes with a permanent marker.

  1. Add 2 volumes of autoclaved H2O for each volume of cell pellet.2. Bring to 1 mM PMSF.3. Sonicate on ice . Wait 30 seconds between each sonication.4. Pellet debris in the microcentrifuge at 13,000 RPM for 5 minutes.5. Dilute with water to OD280 = 3.0. Store at -20°C. These stocks should be good for at least one year.

C: 30% Acrylamide Stock

3) Peel off tape from the bottom of the cassette.

Preparation of Bacterially Expressed Proteins

  1. Add 292 g acrylamide and 8 g piperazine diacrylamide to 700 ml HPLC-grade H2O, under the hood.
  2. Stir to dissolve.
  3. Bring volume to 1 L.
  4. Filter through 0.45 µm pore size filter.
  5. Store at 4°C in the dark.

4) Insert gel into the Mini-Cell so that you can read "10 % BIS-TRIS" when facing the gel. If only running one gel use the square plastic dam to replace the second gel cassette.

  1. Grow a 10 ml bacterial culture to saturation with the appropriate antibiotic. If IPTG induction is needed, transfer 200 µl of the saturated culture to 10 ml of L broth with antibiotic. IPTG may be added at this time, or the cells may be grown to attain exponential growth before adding the IPTG. Add 10 - 100 µl of 1 M IPTG so that the final concentration of IPTG is from 1 - 10 mM. Induce protein synthesis for 1 - 3 hours. The final yield of protein may be affected by the concentration of IPTG, the induction time, and the bacterial concentration.2. Centrifuge at 2,500 RPM for 15 minutes at 4°C. Discard the supernatant.3. Dissolve the pellet in 1 ml of lysate buffer. Bring to 1 mM PMSF by adding 100 mM PMSF stock . This solution may be kept for 1 week at -20°C.4. Add 50 µl of a 10 mg/ml lysozyme solution made in 0.25M Tris-HCl, pH8. Make the lysozyme solution fresh each time. Do not vortex the enzyme. Shake the cells on ice for 15 minutes.5. Transfer the cell solution to a 4 ml Falcon tube and sonicate at setting "4" for about 10 seconds on ice.6. Transfer this lysate to microfuge tubes and centrifuge for 15 minutes on high speed at 4°C.7. At this point, you will need to determine if the antigen is in the supernatant or in the pellet using a Western blot or ELISA.

D: Separating Acrylamide Gel

Below are the solution volumes required to prepare one 9-18% gradient gel. Prepare sufficient volume for the number of gels to be run.

5) Check for tightness of the seal by filling the inner chamber with a small amount of buffer. If a leak is detected reposition or reassemble the chamber.

SDS-PAGE Separating Gels :

Solution

Volume Units

9% gel

18% gel

1.5 M Tris-HCl, pH 8.8

ml

11.5

11.5

20% SDS

ml

0.23

0.23

30% Acrylamide

ml

14

28

TEMED

µl

11.7

11.7

10% APS

µl

117

117

HPLC-grade H2O

ml

20

6

Total

ml

45.8

45.8

6) Remove the comb from the gel. Using a disposable needle tip, remove any residual buffer from the wells.

  1. For 2 gels, mix in an Erlenmeyer flask in the fume hood with constant stirring under vacuum for 15 minutes:6 ml lower gel buffer at room temperature 6 ml 40% acrylamide12 ml double distilled H20Increase the percentage of acrylamide to analyze lower molecular weight products , but continue to use a 10% stacking gel.2. Assemble the gel holder. Wear gloves to clean the plates with methanol or 95% ethanol. Dry with Kimwipes. Assemble in a gel caster in the following order :a). wax paperb). thick plastic platec). thin, flexible plastic spacer plated). white glass platee). Gray plastic side spacerf). Clear glass plateg). thin, flexible plastic spacer plateh). Repeat 4-7, then clamp sides.3. Add 0.12 ml of 10% ammonium persulfate solution and 12µl of TEMED to the acrylamide solution prepared above. The APS solution should not be more than 1 week old.4. Pour the acrylamide solution into the gel holder to about 1 cm above the black mark. This black mark is 3 cm from the top of the plastic holder. Add 1/2 Pasteur pipet full of H20 saturated butanol to each plate.5. Wait at least 45 minutes for polymerization then rinse the top of the gels with distilled H20. These gels may be stored at room temperature for about 1 week in a sealed container with H20 soaked towels to avoid dessication of the gels.6. Clamp in electrophoresis holder and rinse again with H2O, then blot the top of the gel with filter paper to remove excess H2O.

E: 50 ml Equilibration Buffer I

  1. Mix together 18 g urea and 10 ml of 0.5 M Tris-HCl, pH 6.9.
  2. Vortex vigorously.
  3. Add 10 ml of 20% SDS and 200 mg DTT.
  4. Invert gently several times.
  5. Add 15 ml glycerol.
  6. Vortex vigorously .
  7. Add Bromophenol Blue as indicator.

F: 50 ml Equilibration Buffer II

  1. Same as Equilibration Buffer I EXCEPT add 5.0 g Iodoacetamide INSTEAD of DTT.

G: Transfer Buffer

7) Proceed to fill the inner chamber with running buffer so that the buffer level covers the level of the wells. If running reduced samples add .5 mL of NuPAGE Anti-oxidant.

Prepare 10% SDS Stacking Gel:

  1. For 1 L:25 mM Tris-HCl 190 mM glycine 20% methanol

8) Load samples onto gel and pour running buffer into the outer chamber until it covers about half of the cassette.

1.25 ml upper gel buffer at room temperature0.56 ml 40% acrylamide3.2 ml H2O30 µl 10% APS solution10 µl TEMEDLoad the upper gel with a Pasteur pipet and add the gel comb. Watch polymerization about every 3 minutes to see if more upper gel solution needs to be added. It should be completely polymerized within 15 minutes. Remove comb and add Tank Buffer to the top with a funnel. Remove any bubbles at the bottom of the gel. Rinse all wells with tank buffer.

Day One: Sample Preparation

9) Run gels according to the protocol which is applicable:

Preparation of Sample:

A. Tissue Processing

  • To lyse a 5-8 µm tissue section obtained from a paraffin-embedded block:
  1. Place the tissue section in a 1.5 ml Eppendorf tube.
  2. Add xylenes to cover the tissue.
  3. Vortex vigorously for ~15 sec.
  4. Incubate at RT for 5 min.
  5. Vortex again.
  6. Spin down for 3 min to pellet the tissue.
  7. Remove xylenes.
  8. Add 1 ml xylenes.
  9. Vortex 5-10 sec.
  10. Spin down.
  11. Remove xylenes.
  12. Speed vacuum the sample for a few minutes to evaporate the remaining xylene.
  13. Add 400 µl IEF buffer.
  14. Vortex vigorously for 1 min.
  15. Incubate for 5 min at RT.
  16. Vortex vigorously for 1 min.
  17. Spin sample down at 14,000 g for 5-10 min at room temp.
  • To lyse a 5-8 µm tissue section obtained from a polyester-embedded block:

Follow 1-15 above for a paraffin-embedded block section, EXCEPT use 100% EtOH instead of xylenes.

  • To a 5-8 µm tissue section obtained from a frozen block OR a microdissected tissue sample:
  1. Place the tissue section or microdissected tissue sample in a 1.5 ml Eppendorf tube.
  2. Add 400 µl IEF buffer.
  3. Vortex vigorously for 1 min.
  4. Incubate for 5 min. at RT.
  5. Vortex vigorously for 1 min.
  6. Spin sample down at 14,000 g for 5-10 min at room temp.
  • To lyse a paraffin-embedded section on a slide:
  1. Deparaffinize the tissue by immersing the slide into xylenes, twice for 5 min each.
  2. Allow the tissue section to dry.
  3. Add 200 µl of the IEF buffer to the tissue and pipet up and down several times.
  4. Remove the buffer into a microfuge tube.
  5. Scrape the tissue with a razor blade and transfer into the same microfuge tube.
  6. Add 200 µl to the lysates for a total of 400 µl.
  7. Vortex vigorously for 1 min.
  8. Incubate for 5 min at RT.
  9. Vortex vigorously for 1 min.
  10. Spin sample down at 14,000 g for 5-10 min at room temp.
  • To lyse a polyester-embedded section on a slide:
  1. Remove the polyester by immersing the slide into ethanol, twice for 5 min each.
  2. Repeat steps 2-10, directly above, as for a paraffin-embedded section on a slide.
  • To lyse a frozen section on a slide:
    1. Thaw the slide to room temperature.
    2. Immerse the tissue section into xylenes for 1 min.
    3. Repeat steps 2-10, directly above, as for a paraffin-embedded section on a slide.

B. Reswelling

  1. Remove Immobiline Drystrips from -20°C and allow to equilibrate at RT.

    TIP: The pH range of the strip used should be the same as the pH range of Pharmalytes or IPG buffer used in the IEF lysis buffer.

  2. Notch the basic end of the strip to mark each sample.

  3. Load the first sample into Reswelling trays
  4. Place DryStrips gel-side down into each slot.
  5. Remove air bubbles by pressing down with a pipette tip.
  6. Overlay completely with DryStrip Cover fluid .
  7. Repeat for every sample including the MW standard .
  8. If samples are concentrated in one region of the strip, redistribute by pipetting.
  9. Cover the tray with the lid.
  10. Incubate overnight at RT to allow the strips absorb the samples.

10% Bis-Tris Gel with MES Running Buffer

  1. Consider volumes to be loaded: For the 1 well comb, load 10 µl of protein molecular weight markers and 500 µl of sample. Use 30 µl of sample per well for the 10 well comb. If a similar gel is prepared for Coomassie blue or silver staining, additional protein needs to be loaded relative to that used for Western blots. See the Coomasie and Silver Staining protocol for details.2. Add sample buffer to the samples:* Molecular weight markers from Promega are at 0.5 mg/ml. These are diluted 1:2 in H2O and stored at -20°C. Before use, add 20 µl of this dilution to 10 µl of H20 and 10 µl of 4x sample buffer . Molecular weights are listed below.* For human cell extracts add 167 µl of 4x sample buffer to 500 µl of Molt-4 extract before heating. This will dilute the sample buffer to a 1x concentration.* For bacterial cell extracts, dissolve the final antigenic pellet in 500 µl of 2x sample buffer, then dilute 1:10 in additional 2x sample buffer.* For insect cells infected with baculovirus, use a lysate from 3000 cells per lane in a 10 well comb with diluted sample buffer.

Day Two: 1st Dimension

Voltage: 200V constant

# Heat samples at 95°C for 10 minutes before loading on the gel.Electrophoretic Separation of Proteins:

  1. Clean the electrophoresis chamber and the Immobiline strip tray and wipe out with paper towels and Kimwipes to remove mineral oil.
  2. Place the tray on top of 50 ml DryStrip Cover fluid.
  3. Remove strips.
  4. Place on Whatman paper gel-side up.

    TIP: Placing the strips gel-side down might result in protein loss and gel damage.

  5. Leave for ~1 min.

  6. Place into the electrophoresis chamber gel-side up.
  7. Arrange the strips so that their edges are in one line.

    TIP: Time is important to prevent crystallization.

  8. Wet the pre-made IEF electrode strips with HPLC-grade H2O.

  9. Dry slightly between two pieces of Whatman paper.
  10. Place 2 buffer strips on both edges of the strips and perpendicular to them, covering the top of the bromophenol blue on each side.
  11. Make sure that the square end of each strip is at the cathode end and the pointed end is at the anode end and also that the anode and cathode electrode ridges are in the correct orientation.
  12. Overlay liberally with DryStrip Cover fluid between the immobilon strips and outside the electrodes.
  13. Electrophorese for 36-48 hrs, using the following sequence of settings:

    Voltage Amps Wattage Time

    1

    500 V 100 mA 33 W 0.05 hrs

    2

    500 V 110 mA 70 W 1 hr

    3

    3500 V 141 mA 32 W 5 hrs

    4

    3500 V 70 mA 38 W Until stopped
  14. The bromophenol blue should be seen migrating towards the anode within at least 1 hr. By next day, the strips should be colorless.

  15. If by the next day the bromophenol blue has not disappeared, running can be paused and the electrode strips can be replaced from whichever side. Continue running until the dye has disappeared.

Expected Current: Start: 110-125 mA per gel; End: 70-80 mA per gel

For 1 gel, electrophorese initially at 20 mA constant current. When the dye passed through the stacking gel, increase to 30 mA. Continue electrophoresis until the dye has run off bottom of the gel . Alternatively, an overnight electrophoresis may be performed by running the gel until the dye is half through the gel on the above settings, and then decreasing the power to 5 mA overnight. In the latter case, the 31kD molecular weight marker should be at the bottom of the gel in the morning. Disassemble the apparatus, and discard the stacking gel. Notch the bottom right corners of the separating gel and a precut nitrocellulose membrane. Place the membrane on top of the gel, and peel the gel off of the plate.

Day Three: 2nd Dimension

Approx. Run Time: 35 minutes

For 2 gels, follow above procedure but double the current settings.

A: Prepare Apparatus

  1. Wash and scrub plates very well in soap and hot water.
  2. Rinse in diH2O.
  3. Leave the plates to air dry or wipe with methanol-soaked Kimwipes.
  4. Order plates in Protean-II Multi-Gel casting Chamber as follows:

    Bottom of chamber
    Small plate, 20 cm
    Spacers, 20 cm x 1 mm
    Eared plates, 20 cm
    Spacers, 20 cm x 1 mm
    Large plate, 20 cm
    Meylar sheet
    Repeat as needed.
  5. Fill the chamber with acrylic blocks.

  6. Tighten the screws.
  7. Tape the edges of the chamber to prevent leakage.

B: Prepare Gradient Acrylamide Gel

  1. Add 9% gel solution to the center compartment of the distributor and 18% gel to the peripheral compartment .
  2. Start the magnetic stirrer in the mixing chamber.
  3. Remove air bubbles from the tubing by opening the valve slowly.
  4. Allow the tubing to fill with gel solution, then close the valve.
  5. Turn on the stirrer.

    TIP: The stir bar should be between the openings between the mixing and reservoir compartments, but not on top of them.

  6. Open the valve between the mixing and the reservoir chambers .

  7. Make sure the 18% solution is flowing into the mixing compartment.
  8. Open the valve to start the flow of the acrylamide solution into the Protean chamber.
  9. Allow reasonable flow. Fast flow results in loss of a gradient, whereas a slow flow results in polymerization of the solution in the tubing. In addition, the rate of flow changes with time due to the change of pressure. However, the chamber should be filled in at least 10 minutes.
  10. Stop when the gel has reached 0.5 cm from the top of the glass plates.
  11. Overlay the gels carefully with HPLC-grade H2O using a syringe.
  12. Cover with Saran Wrap.
  13. Allow to polymerize overnight.

10% Bis-Tris Gel with MOPS Running Buffer

Electrophoretic Transfer for Dry Blots :

Day Four: 2nd Dimension

Voltage: 200 V constant

Precut three pieces of Whatman 3MM filter paper to the size of the gel. Saturate one filter with working solutions A1, A2, or C, respectively. On the bottom electrode, place in the following order filter A1, then filter A2, the nitrocellulose membrane, the polyacrylamide gel, and finally filter C. Remove any bubbles after each step. Place the top electrode on the stack. Begin transfer at 10-12 constant volts. Regulate voltage to keep the current at 105 mA. Do not allow voltage to exceed 15 V. The minimum transfer time is 40 minutes, the maximum is 60 minutes. You will obtain poor transfer if more than 60 minutes is used.

  1. Remove strips.
  2. Place on Whatman paper, gel-side up, for 1 min.
  3. Equilibrate in Equilibration Buffer I for 10-15 min.
  4. Set-up gels in electrophoresis chamber .
  5. Make sure there is no leakage.
  6. Equilibrate strips in Equilibration Buffer II.
  7. Remove strips.
  8. Place on Whatman paper one by one, gel-side up.
  9. Identify notches.
  10. Cut ~one inch from both sides.
  11. Place gels with basic side closer to the anode and the acidic side closer to the cathode to be consistent.
  12. Run gels at 40 mA/gel for 15 min or until dye front is about an inch into the gel.
  13. Turn down voltage and run at 65-70 V constant overnight. .

Expected Current: Start: 110-115 mA per gel; End: 60-70 mA per gel

Electrophoretic Transfer For Wet Blots:

Day Five: Transfer and Sequencing of Proteins

Approx. Run Time: 50 minutes

  1. Soak the gel and the nitrocellulose membrane in wet blot transfer buffer for a minimum of 15 minutes to remove electrophoresis salts and detergents.2. For each gel, saturate two fiber pads and two precut Whatman 3MM filter papers in transfer buffer.3. Assemble on the gray side of a cassette in the following order:1 fiber pad1 Whatman 3MM filter paperGelNitrocellulose membrane1 Whatman 3MM filter paper1 fiber pad4. Repeat with a second cassette for the second gel, if needed.5. Insert the cassette into the electrode module. Be sure to check the direction so that the transfer is from the gel to the membrane. The gray side of the cassette should be next to the gray side of the electrode module .6. Place a stir bar and a Bio-Ice cooling unit in the buffer tank. Place the electrode module in the buffer tank.7. Fill the tank with wet blot transfer buffer to the bottom edge of the top row of holes on the cassette. Place the buffer tank on a magnetic stir plate and stir at medium speed.8. Attach electrodes and electrophorese at 100 V for 1 hour, or 30 V overnight.

A: Transfer onto PVDF or Nitrocellulose Membranes

  1. Soak gel in transfer buffer for 5-10 min.
  2. Soak pre-cut transfer membrane and Whatman paper in transfer buffer.
  3. Wet the transfer apparatus with transfer buffer.
  4. Place 3-4 Whatman papers on bottom of the transfer apparatus.
  5. Place membrane on top and remove air bubbles.
  6. Place gel on top of the membrane and remove air bubbles.
  7. Place 3-4 Whatman papers on top of the gel and remove any air bubbles.
  8. Transfer for 3 hrs at no more than 25V . Transfer for 5 hrs if more than 5-6 gels.

B: Gel Staining

See Staining Protocols

C: Excision of Protein Spots

TIP: Work under the hood to prevent the gel from being exposed to too much air, thus avoiding contamination with keratins

4-12% Bis-Tris Gel with MES Running Buffer

Detection of Antigen on Membranes:

  1. Xerox the gel and assign the spot to be sequenced.
  2. 皇家赌场号hj85,Cut out the protein spot for sequencing with a pipette tip.
  3. Remove to a microfuge tube.
  4. Chop up the gel piece with pipette tip.
  5. Add a solution of 50% methanol/10% acetic acid to the gel pieces.
  6. Incubate for 30 min.
  7. Spin down.
  8. Remove the supernatant.
  9. The sample is ready for Mass Spectrometry Sequencing.

Voltage: 200V constant

  1. Stain the membrane with 0.1% fast green for 1 minute. Rinse four times with H20. Cut away the molecular weight marker lane and shake it in TBST for 30 minutes . The rest of the membrane may be sliced in strips, if needed. For a 10 well comb, each well may be cur in half. Notch the left bottom edge of all strips for orientation purposes.2. Block the membranes in a 5% non-fat milk-TBST solution for at least 30 minutes while shaking. For 3 mm strips use about 1 ml of TBST-milk. Membranes may be stored in TBST at 4°C for up to 1 week.3. Remove the blocking solution. Rinse membranes in TBST once.4. Add 500 µl for thin sections, or 5 ml for an entire membrane, of serum diluted in 5% milk-TBST. A typical serum dilution is 10-2. Incubate at room temperature for 30 minutes while shaking.5. Wash 4 times for five minutes per wash in TBST at room temperature.6. Add 500 µl of diluted alkaline-phosphatase conjugated secondary antibody and incubate for 30 minutes at room temperature with shaking. .7. Wash 4 times for five minutes per wash with TBST at room temperature.8. Add 500 µl of BCIP/NBT substrate . This solution should be yellow, not brown. Wait about 15 minutes for bands to appear. Watch carefully since the reaction times will vary. Rinse with water to stop developing. While the membranes are developing, stain the molecular weight marker lane with 0.1% Amido Black solution for 1 minute, remove this membrane and immediately add to Amido Black destain for about 10 seconds before rinsing extensively in H20.

Expected Current: Start: 110-125 mA per gel; End: 70-80 mA per gel

Photograph the membranes with Polaroid Type 57 film.

Approx. Run Time: 35 minutes

Focus with the overhead and focusing lights on, and the shutter and f-stop wide open . Close the shutter. Photograph using only the room lights at f8, 1/60 second exposure.

4-12% Bis-Tris Gel with MOPS Running Buffer

RECIPES

Voltage: 200V constant

Lysate Buffer For 100 ml, add 2 ml of 5 M NaCl, 5 ml of 1 M Tris-HCl stock at the appropriate pH, 2 ml 0.5 M EDTA and Q.S. with distilled water. Autoclave.

Expected Current: Start: 110-115 mA per gel; End 70-80 mA per gel

5% Nonfat Dry MilkPrepare using Carnation Nonfat Dry Milk in TBST.

Approx. Run Time: 50 minutes

10% Ammonium Persulfate Solution0.12 g APS1.2 ml H2O)Keep for no more than week week in the refrigerator.

10) When gel is complete, shut off power, disconnect electrodes and remove gel from Mini-Cell

TBST

11) Separate each of the three bonded sides by prying them open with a screw driver. Using a razor blade cut the sides of the gel. The thick edge at the bottom of the gel should be cut off.

Chemical For 4 liters For 500 mlTris 4.84 g 0.61 g TrisNaCl 35.06 g 4.38 g NaClNaN3 2.0 g 500 µl 10% NaN3Tween-20 2.0 ml 250 µl Tween-20Adjust pH to 8.0 with HCl. QS with water and store at 4°C.

Sample Preparation

Tank Buffer

1) Prepare sample buffer by mixing 50 µL of NuPAGE Sample Buffer, 30 µL D.I. water, and 20µL of reducing agent.

Chemical For 4 liters For 500 mlTris 12.11g 1.51 g TrisGlycine 57.6 g 7.2 gSDS 4.0 g 17.5 ml 10% SDSQS with H2O. pH should be 8.3 but adjusting is not needed. Store at 4°C

2) Add sample buffer in a 1:1 ratio to sample

Transfer Buffers:

3) Heat mixture of sample buffer and sample for 5 minutes in boiling water bath.

Wet blot transfer buffer .9.08 g Tris 43.24 g glycine 600 ml methanol QS to 3 L with water.Dry blot transfer buffers

4) Heat See-Blue standard for 3 minutes in boiling water bath.

Stock Solution A1 36.3 g Tris, dissolved in double distilled water and QS to 100 ml Store at room temperature. Stock Solution A2 3.63 g Tris, dissolved in double distilled water and QS to 100 ml Store at 4°C. Stock Solution C 3.63 g Tris, 5.2 g aminohexane, dissolved in double distilled water and qs to to 100 ml. Store at 4°C.

Dry Blot Working Solutions 7 ml H20 2 ml Methanol 1 ml stock solution of A1, or A2 or C Store at 4°C.

Lower Gel Buffer For 100 ml: 18.17 g Tris 4 ml 10% SDS Adjust pH to 8.8 with HCl. QS to 100 ml with water. Store at room temperature.

Upper Gel Buffer 0.5 M Tris-HCl, 0.4% SDS, pH 6.8)For 100 ml: 6.06 g Tris 4 ml 10% SDS Adjust pH to 6.8 with HCl. QS to 100 ml with water. Store at room temperature.

4x Sample Buffer 8 ml of stock sample buffer 2 ml of concentrated 2-mercaptoethanol Transfer to 1 ml aliquots and store at -20°C.

2x Sample Buffer .Prepare in fume hood! 2.5 ml 0.5 M Tris-HCl, pH6.8 and 4.0 ml of 10% SDS 2.0 ml glycerol 1.0 ml concentrated 2-mercaptoethanol 0.4 mg bromophenol blue QS to 10 ml with water. Transfer to 1 ml aliquots and store at -20°C.

Stock Sample Buffer 3.03 g Tris 8.0 g SDS 40 ml glycerol Adjust pH to 6.8 with HCl. QS to 80 ml with water. Store at room temperature.

0.1% Fast Green 0.2 g Fast Green 20 ml acetic acid 40 ml methanol 140 ml double distilled water.

0.1% Amido Black 0.2 g Buffalo Black 90 ml methanol 20 ml acetic acid 90 ml double distilled water.

Amido Black Destain 225 ml methanol 10 ml acetic acid QS to 500 ml with double distilled water.

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