Polyacrylamide Gel Electrophoresis

BIOLOGY 371

INDEPENDENT RESEARCH

WEEK 11

Polyacrylamide Gel Electrophoresis - Native Molecular Size

1. Prepare samples of crude homogenate, 40 - 65% pellet and "purified IDH", ie., concentrated prep off Matrex Red column.
2. Set up BioRad MiniGel unit with 7.5% or 12% polyacrylamide gels.

Do not use SDS at any step.

3. Run samples. Stain 1/2 the gel with Coomassie Blue and 1/2 for IDH activity [or run two gels].
4. Estimate molecular size and purity of IDH.

IDH Activity In Polyacrylamide Gels

Objectives

1. To determine relative purity of our preparation.
3. To identify IDH activity in gels associated with specific protein bands.
2. To estimate the molecular size of IDH.

Procedure

Use 12% polyacrylamide gels and BioRad Pre-stained "high range" molecular weight markers.

SDS Polyacrylamide Gel Electrophoresis

WARNING: Unpolymerized acrylamide is extremely toxic. Wear gloves and never pipet by mouth.

Stock Solutions

A. Acrylamide/bis
Acrylamide 29.9 gm
N'N'-bis-methylene-acrylamide 0.8 gm
Make to 100 mL with water.
Store at 4° C in the dark [30 days max].

B. 1.5 M Tris-HCl, pH 8.8
Tris base 27.23 gm
80 mL water
Adjust to pH 8.8 with 6N HCl.
Make to 150 mL with water.
Store at 4° C.

C. 0.5 M Tris-HCl, pH 6.8
Tris base 6.1 gm
80 mL water
Adjust to pH 6.8 with 6N HCl.
Make to 100 mL with water.
Store at 4° C.

D. 10% SDS
Add 10 gm SDS in 90 mL water.
Stir gently to dissolve.
Make to 100 mL

E. Sample Buffer / SDS Reducing Buffer
Water 3.8 mL
0.5 M Tris-HCl, pH 6.8 1.0 mL
Glycerol 0.8 mL
10% SDS 1.6 mL
2-Mercaptoethanol 0.4 mL
1% Bromophenol Blue 0.4 mL
Dilute sample 1:4 and heat at 95° C for 4 minutes.

F. 5X Running Buffer, pH 8.3
Tris base 9.0 gm
Glycine 43.2 gm
SDS 3 gm
Make to 600 mL with water.
Store at 4° C.
Dilute 60 mL of 5X buffer with 240 mL water for one run.

Separating Gel - 0.375 M TRIS, pH 8.8

Reagent 12% Gel a 7.5% Gel b

Distilled water 3.35 mL 4.85 mL

1.5M Tris-HCl, pH 8.8 2.5 mL 2.5 mL

10% SDS Stock 100 µl 100 µl

Acrylamide/Bis Stock 4.0 mL 2.5 mL

10% Ammonium Persulfate* 50 µl 50 µl

TEMED 5 µl 5 µl

* Prepare fresh solution. Dissolve 100 mg of APS in 1 mL of water.
Prepare any volume by using multiples of 10 mL recipes.
Gel with 1.0 mm spacer will require about 5.6 mL
a. For SDS-treated proteins in the molecular weight range of 10 - 100 kDa.
b. For SDS-treated proteins in the molecular weight range of 40 - 250 kDa.

Staining for Proteins

Stain about 30 minutes at room temperature.
0.1% Coomassie Blue R250
40 % Methanol
10% Acetic acid
Destain with several changes of destaining solution.
40 mL Methanol
75 mL Acetic acid
To 1 Litre

Staining for NADP⁺ - IDH

1. 0.06 M Tris buffer: 1.256 gm Tris - HCl, pH 8.2
0.465 gm Tris base
0.153 gm MgCl₂[anhydrous], 8 mM*
Water to 200 mL
2. To 100 mL, add: 2.7 mg NADP⁺, 0.35 mM
19.6 mg NB-Tetrazolium, 2.4 mM
2.1 mg Phenazine Ethosulfate, 0.6 mM
Note: This is light sensitive - cover with foil.
3. Staining: Add 20 mg of DL-isocitrate.
4. Place in dark for 1 hour [or more].

*Concentrations of Mg²⁺ greater than 10 mM are inhibitory to the electron transferring ability of PMS

From: Rothe, G. M., 1994. Electrophoresis of Enzymes - Laboratory Methods. Springer Verlag, Berlin, p 231.

Protein Standards

At least four proteins of known molecular weight should be chosen to construct a standard curve. They should cover a range of values such that the molecular weight of your unknown samples will fall well within that range. Some useful proteins to use as standards are:

Protein Molecular Weight & Subunit Weight
Phosphorylase A 92,500 & 92,500
BSA 67,000 & 67,000
Glutamate dehydrogenase 220,000 & 53,000
Avian albumin 45,000 & 45,000
Aldolase 158,000 & 40,000
Chymotrypsinogen A 25,000 & 25,000

Example:
Estimation of native size of cauliflower IDH
Standard Mol Weight / Log MW / Migration [mm]
Myosin 206,000 / 5.3139 / 4
b -galactosidase 117,000 / 5.0682 / 8
BSA 89,000 / 4.9494 / 19
Ovalbumin 47,000 / 4.6721 / 38
IDH 19.5

Corr = -0.96
IDH: 17 mm --------> 91,165 Da

Figure 18. Activity of Chlamydomonas IDH in an acrylamide gel. IDH was purified using Matrex Red chromatography. The estimated molecular size of the upper band is 180 kDa; that of the lower band is 91 kDa [Williamson and A Malcolm Campbell, August, 1997].

Figure 19. SDS acrylamide gel stained with Coomassie Blue R250.

Return to Schedule

Reagent	12% Gel a	7.5% Gel b
Distilled water	3.35 mL	4.85 mL
1.5M Tris-HCl, pH 8.8	2.5 mL	2.5 mL
10% SDS Stock	100 µl	100 µl
Acrylamide/Bis Stock	4.0 mL	2.5 mL
10% Ammonium Persulfate*	50 µl	50 µl
TEMED	5 µl	5 µl