6x loading dye recipe buffer tube

16-10-2016, 10:10

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Materials

BCA protein assay (Pierce Biotech. cat. #23225)
Small-PROTEAN II gel apparatus (Biorad)
Costar gel-loading tips (Krackler Scientific, cat. #MN520R-LRS)
Prestained SDS-Page broadrange molecular weight standard (NEB, cat. #P7708S)
Trans-blot Semi-Dry Transfer cell (Biorad)
Immobilon-P membrane (Millipore, cat. #IPV00010)
heat-sealable bags (Kapak, cat. #TRS-95250)
Kodak Biomax light autoradiagraphy film, 13 x 18 cm (Perkin Elmer Existence Sciences, cat. #868-9358)
Radtape (Diversified Biotech, cat. #RAD-10)

Reagents

TEMED (Sigma)
Tween-20 (Sigma)
Cost Chopper non-fat dry milk
Isopropanol (Sigma)

Gel Buffers

Acrylamide solution (30% acrylamide/.8% bis-acrylamide)

-dissolve 30g acrylamide (FW=71.08), .8g bis-acrylamide (FW=154.17) inside a total amount of 100ml water. Filter through .2um filter.

4X Running Gel Buffer (1.5M Tris, pH 8.8)

-dissolve 36.3g Tris Base (FW=121.1) in 150ml water. Adapt to pH 8.8. Add water to total amount of 200ml. Filter through .2um filter.

4X Stacking Gel Buffer (.5M Tris, pH 6.8)

-dissolve 12.1g Tris Base (FW=121.1) in 150ml water. Adapt to pH 6.8. Add water to total amount of 200ml. Filter through .2um filter.

10% SDS

-dissolve 10g SDS (FW=288.38) inside a total amount of 100ml water.

10% Ammonium Persulfate

-dissolve 1.0g APS (FW=228.2) inside a total amount of 10ml water. Store at -20C in 50-100ul aliquots.

1M Tris, pH 6.8

-dissolve 12.1g Tris Base (FW=121.1) in 80ml water. Adapt to pH 6.8. Add water to total amount of 100ml. Filter through .2um filter.

6X SDS Sample Buffer (.375M Tris pH 6.8, 12% SDS, 60% glycerol, .6M DTT, .06% bromophenol blue)

-combine 3.75ml 1M Tris-Cl, pH 6.8, 6ml glycerol, 1.2g SDS (FW=288.38), .93g DTT (FW=154.2), 6mg bromophenol blue. Add water to total amount of 10ml. Store at -20C in .5ml aliquots.

2X SDS Sample Buffer (.125M Tris pH 6.8, 4% SDS, 20% glycerol, .2M DTT, .02% bromophenol blue)

-combine 2.5ml 4X Tris pH 6.8 (stacking gel buffer), 2ml glycerol, 4ml 10% SDS (FW=288.38), .31g DTT (FW=154.2), 2mg bromophenol blue. Add water to total amount of 10ml. Store at -20C in .5ml aliquots.

1X SDS Sample Buffer (.0625M Tris pH 6.8, 2% SDS, 10% glycerol, .1M DTT, .01% bromophenol blue)

5X Electrophoresis Buffer (.125M Tris, .96M glycine, .5% SDS)

-dissolve 30.3g Tris Base (FW=121.1), 144.1g glycine (FW=75.07), and 10g SDS (FW=288.38) inside a total amount of 2L water.

1X Electrophoresis Buffer (.025M Tris, .192M glycine, .1% SDS)

-add 200ml 5X electrophoresis buffer to 800ml water.

Western Blot Buffers

Transfer Buffer (.048M Tris, .039M glycine, 20% methanol, .00375% SDS)

-dissolve 11.64g Tris Base (FW=121.1), 5.86g glycine (FW=75.07) in

1500ml water. Add .750ml 10% SDS (FW=288.38). Add 400ml methanol. Add water to total amount of 2L. No pH adjustment necessary.

10X TBST Wash Buffer (.1M Tris HCl, 1.5M NaCl, .5% Tween-20)

-dissolve 31.52g Tris HCl (FW=157.6), 175.32g NaCl (FW=58.44) in

1900ml water. Add 10ml Tween-20. Adapt to pH 7.2. Add water to total amount of 2L.

1X TBST Wash Buffer (.01M Tris HCl, .15M NaCl, .05% Tween-20)

-add 100ml 10X TBST to 900ml water.

5% milk Blocking Buffer

-Dissolve 5g milk inside a total amount of 100ml 1X TBST.

Method

Determine cell phone number or protein concentration to become loaded onto gels and just what volume to load per well. Note max volumes for various comb configurations.

Usually add sample to Tris or PBS after which add sample buffer (2 ul sample + 18 ul PBS + 3.3 ul 6X sample buffer).

Sonicate cells briefly on ice to homogenize. Purified protein samples don't need to be sonicated.

Preparing resolving and stacking gels (for BioRad Small-PROTEAN II): Make certain glass plates are clean. Use Sparkleen or Alconox powder to wash plates. Rinse with sterilized water after which 95% EtOH. Wipe dry with KimWipes. Pick a comb and spacer (ex. .75 mm). Place spacers among the interior and outer glass plates. Make certain the spacers are flush using the plates. Insert plates in to the clamping set up and tighten screws. Vacuum grease may be used around the casting stand gaskets to avoid buffer leakage. Snap set up in to the casting stand. Prepare resolving gel. Pour gel and overlay with 1X Electrophoresis Buffer or isopropanol (use 3.5ml resolving gel when utilizing .75mm spacers and seven.0ml gel for 1.5mm spacers, this can create a gel approximately. 3.25 x 2). Locate a division between your overlay and resolving gel, what this means is the gel is polymerized. Pour off overlay, use thin strips of Whatman paper to get rid of any excess. Pour stacking gel and insert comb. Remove comb when stacking gel is polymerized. Make use of a syringe along with a 12 gauge needle to clean stacking gel wells. Snap clamp set up into electrode set up. Vacuum grease may be used on electrode set up gaskets to avoid buffer leakage.

Boil samples a few minutes at 100°C, after which spin tubes at 13,000rpm for 1min. in RT micro centrifuge. Load samples using Costar Gel-Loading Tips.

Place electrode set up with loaded samples into an electrophoresis chamber. Fill the electrode set up carefully with 1X Electrophoresis Buffer . Fill the electrophoresis chamber with 1X Electrophoresis Buffer . Run gels at 100V for stacking gel, 100-150V for resolving gel (can increase to 200V for resolving gel if running on ice).

To transfer small- gels to Immobilon-P membrane while using BioRad Semi-Dry Transfer Cell: Remove gels from glass plates. Cut stacking gel away and discard. Incubate gels in Transfer Buffer for twenty to forty min. Eliminate 6 bits of Whatman paper and 1 bit of Immobilon-P membrane for every gel that's being transferred. They must be 3.25 x 2. Incubate Immobilon-P membrane in Methanol for ten to twenty seconds. Wet Immobilon-P membrane and Whatman paper in Transfer Buffer for five to ten minutes before assembling the gel sandwich based on the BioRad Reference Guide. Transfer 1 small-gel for 15 min. at 15 V, max current. Transfer 2 small-gels for 30 min at 15 V, max current. Make certain that pre-stained MW markers transfer to Immobilon-P membrane.

Western Blot:

Block membrane in 20ml blocking buffer overnight at 4°C or 1h at 70 degrees on rocker inside a seal-a-meal bag.
Dilute Ab in blocking buffer (1:1000 for serum or 5ug / 10ml for purified Ab). Incubate blots with Ab for 1h at rom temperature or overnight at 4°C on rocker inside a seal-a-meal bag.
Wash blots with 50ml 1X TBST on orbital shaker 3 occasions, a few minutes each.
Dilute HRP conjugated Ab 1:10,000. Incubate blots with secondary Ab for 1h at 70 degrees or overnight at 4°C on rocker inside a seal-a-meal bag.
Wash blots with 50ml 1X TBST on orbital shaker 3 occasions, a few minutes each.
Add 5ml chemiluminescent substrate to every blot, incubate for five minutes. Place blots among transparency film. Push out air bubbles. Develop Western blot. Perform a one minute contact with check signal intensity. Continue longer or shorter exposures when needed.
(Max. exposure length = 60 min. Min exposure length = 1 sec.)
For top backgrounds, wash in TBST then re-develop.