SDS-PAGE/ Western Blotting

Experiment:
Date:
Aim of expt:
Samples:
1.

7.

2.

8.

3.

9.

4.

10.

5.

11.

6.

12.

Procedure:
1. Thaw ______________________________ on ice 15min
2. Prepare ______________ml _____________ lysis buffer. Chill on ice.
3. Add ___________ lysis buffer per sample.
4. Incubate: Ice, 15min
5. Adherent cells on plate: Harvest lysates into microfuge tubes
Cell pellet: Keep in microfuge tube.
6. Rotate: 4C, 30min
7. Spin: Max speed, 4C, 15min
8. Save supernatant for protein estimation
9. Protein estimation: ______________
Blank: Water
Standard: BSA
Sample: 2ul in 500ul

Incubate:
Measure absorbance at OD ______nm
10. Normalise lysates for gel
Volume of lysates needed =
Composition= __________lysate + __________6X + __________Bmer
Sr
Sample
Number

Conc (ug/ul)

Lysate Vol

Water/Lysis buf

11. Controls:
1.
2.
3.
4.
5.
12. Boil: 100C, 5 min
13. Cool, Quick spin
Gel Loading:
1. % of gel and thickness used:
2. Number of wells:
3. Sample volume, Marker volume:
4. Voltage run, Time run:
5. Run length:
6. Sample order:
1

Transfer:
1. Membrane used:
2. Transfer number #:

10

11

12

13

14

15

3. Amperes run, Time run:

Ponceau:
Stain for 2 with rocking, destain with distilled water.
Result:
Blocking: Rotate,
Blotting:
1. Wash membrane 4 times with 1XTBST, 5 at RT.
2. Primary antibody- rotate,
Company:
Isotype:
Concentration:
# Usage:
3. Wash membrane 4 times with 1XTBST, Once with 1XTBS, 5 at RT.
4. Secondary antibody rotate,
Company:
Isotype:
Concentration:
# Usage:
5. Wash membrane 4 times with 1XTBST, Once with 1XTBS, 5 at RT.
6. Scan IR: