Bitesize Bio

SDS-PAGE Cheat Sheet

Essential buffer and gel recipes for your laboratory.

See all of Bitesize Bio's

handy downloadables here.
 SDS-PAGE Cheat Sheet

SDS-PAGE Buffer Recipes

Table 1. Laemmli buffer recipe

Amount for: 2x Amount for: 3x Amount for: 5x

Component
stock stock stock

SDS 4% w/v 6% w/v 8% w/v

BME 10% v/v 15% v/v 25% v/v

Glycerol 20% v/v OR 25.2% 30% v/v OR 37.8% 50% v/v OR 63%
w/v w/v w/v

Tris–HCl, pH 6.8 125mM 187.5mM 312.5mM

Bromophenol 0.005% w/v 0.0075% w/v 0.0125% w/v

blue

Store at 4 °C and use within one year.

Table 2. 10x SDS-PAGE running buffer recipe

Component Amount for: 10x stock

Tris 30.3 g

Glycine 144 g

SDS 10.0 g

H2O 1000 mL

Dilute 100 mL of 10x running buffer stock into 900 mL of H2O to prepare 1x stock.
 SDS-PAGE Cheat Sheet

SDS-PAGE Gel Recipe and Properties

Table 3. SDS-PAGE gel recipe for X% acrylamide gels

Component Amount for X% resolving gel Amount for stacking gel

Acrylamide, 30% (0.5 x X) mL 1.98 mL

Tris, 0.5 M, pH 6.8 0 mL 3.78 mL

Tris, 1.5 M, pH 8.8 3.75 mL 0 mL

SDS, 10% w/v 150 µL 150 µL

H2O 11.02 - (0.5 x X) mL 9 mL

TEMED 7.5 µL 15 µL

APS, 10% w/v 75 µL 75 µL

Total volume 15 mL 15 mL

Wrap gels in damp tissue paper and seal with clingfilm. Store at 4 °C and use within
several weeks.

Table 4. Well volumes of gels prepard using 5-, 10-, and 15-toothed combs

№ of wells 0.75-mm thick gel 1.00-mm thick gel 1.50-mm thick gel

5 70 µL 105 µL 166 µL

10 33 µL 44 µL 66 µL

15 20 µL 36 µL 40 µL

Table 5. Protein sizes that gels can resolve when prepared at 8–20% acrylamide

Size of protein, kDa % Acrylamide in resolving gel

4-40 20

12-45 15

10-70 12.5

15-100 10

25-200 8
 SDS-PAGE Cheat Sheet

SDS-PAGE Gel Casting Protocol

1. Gather Your Equipment

Gather casting equipment and two 50-mL beakers. Label these “R” and “S”.

2. Assemble the Casting Apparatus

Wipe glass sandwich plates clean with industrial methylated spirit (IMS) or ethanol.
Assemble such that the thin glass plate is at the front, the thick glass plate with ridges is
at the back, and the ridges form a narrow gap between the two plates.

3. Pour the Resolving Gel

Pour out all the ingredients for both gels, except the 10% APS and TEMED, into the
corresponding beakers. Seal the one that contains the stacking gel ingredients.

4. Polymerize the Resolving Gel

Add the 10% APS and then the TEMED to the resolving gel ingredients and mix gently.
Pour the mixture immediately into the glass sandwich plates. Leave approximately ~2.5
cm between the meniscus and the top of the front plate.

5. Protect Resolving Gel with Isopropanol

Add isopropanol on top of the unpolymerized resolving gel. Allow ~45 minutes for the
gel to polymerize. Check that the gel has polymerized by tilting the casting equipment.
The top of the resolving gel will stay parallel to the ground when set!

6. Remove Isopropanol
Pour away the isopropanol and wick any excess away using tissue paper. Wash with
distilled water to remove any residual isopropanol.

7. Polymerize the Stacking Gel

Add the 10% APS and TEMED to the stacking gel ingredients, mix, and pour immediately
on top of the polymerized resolving gel. Fill the glass sandwich plates to the top.

8. Insert Sample Comb

Insert a comb into the unpolymerized stacking gel and allow it to set.

9. Remove Sample Comb

Carefully remove the comb in a vertical line to prevent wiggly and collapsed wells.

10. Use or Store Gels

Use gels right away or store at 4 °C and use within several weeks.