Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Standard Operating Procedure: Ultra Hair Protein Additive
Batch Record Number 1214
Recordkeeping Standards:
1.1
Each time a boxed section appears in the right hand margin of the text, one operator
must date and initial that step to certify that they performed it, and another operator
must date and initial to certify that the step was verified. An operator may not verify
their own work at any step of the procedure.
1.2
Only black ball point pen may be used to complete batch records.
1.3
Each operator will use a unique set of initials as identification on batch records.
1.4
All entries on batch records must be legible.
1.5
Errors on an entry must be crossed out with a single line through the center of the entry.
Operators must initial and date each errant entry, and write the replacement next to the
struck-out entry.
1.6
Dates shall be written in the following format: 080208 for August 2, 2008. Do not use
other formats such as 8-2-08 or 8/2/08.
1.7
Time shall be recorded using a 24-hour clock. (8:00 p.m. shall be written as 20:00)
1.8
Complete the comments section of batch record. Included any and all deviations from
the protocol, abnormalities or retests performed. All comments should refer to this
specific step or steps involved as well as specific details of the event along with initials
of the operator observing the event. This also includes nay abnormal readings obtained
from measurements of any kind (e.g., pH meter, spectrophotometer).
Batch Record Table of Contents
Section 1 – Set Up
Section 2 – Buffer Preparation
Section 3 – QC Assay of G25 Column
Note: Section 3 not included as part of lab exercise;
results only included in case discussion
Section 4 – G50 Size Exclusion Column
Section 5 – Ion Exchange Column
Note: Sections 4, 5, and 6 are possible additions to the
batch record depending on technician results in the
laboratory
Section 6 – SDS-PAGE Analysis of Column
Fractions
Section 7 – Comments
1
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
BATCH RECORD NUMBER 1214
Preparation for Purification of UltraHair Protein Additive
Section 1 – Set Up (page 1 of 2)
Step
Instructions
Performed by
Date
Initial
1.00
Put on appropriate safety equipment (lab coat, safety
glasses, gloves)
1.10
Note: if protocol is interrupted and student must leave
immediate area, remove lab coat, gloves, and safety
glasses and put on fresh gloves before restarting.
Obtain all labware needed:
1.20
5 ml plastic disposable pipette with glass wool, tubing and
pinch clamp (column)
Pasteur pipettes and bulb
1.5 ml microfuge tubes
Ring stand and clamp
Small beaker for waste collection
Rack to hold microfuge tubes
Poly-prep chromatography column (Bio-Rad)
Mini-Protean II electrophoresis equipment (Bio-Rad) (or
similar electrophoresis equipment)
Power supply
Micropipettor and tips
Microfuge tubes
Gel drying kit (frame, cellophane sheets, and soaking
solution)
Boiling water bath or heat block
Single edge razor blade
Obtain all chemicals needed:
0.1M potassium phosphate buffer (pH 7.0)
G50 resin (8 ml volume of settled resin/column), swollen
overnight in 0.1M potassium phosphate buffer (pH
7.0)
Batch 0298/12 UltraHair Protein Additive (protein
mixture)
1.0M KCL, 0.1M potassium phosphate buffer, pH 7.0
SP-Sepharose (1.5 ml volume of settled resin/column)
SDS sample buffer
SDS-PAGE running buffer
4-20% preformed polyacrylamide gels, 10-well combs
Molecular Weight standards (NOVEX See-Blue PreStained Standards)
Coomassie Blue stain and destain
2
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 1 (page 2 of 2)
Step
Instructions
Performed by
Date
Initial
1.30
Ensure lab bench or work surface is clear of all items not
pertaining to protocol and has been decontaminated.
Wash solution used:
Time/Date cleaned:
Surface is completely dry:
1.40
yes
no
Check that all scales, stir plates and micropipettes to be
used are in working order. List all equipment to be used
below. Include manufacturer, model number, and
calibration date. (“Performed by” and “verification by”
required for each item.)
3
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 2 – Buffer Preparation (page 1 of 2)
Step
Instructions
Performed by
Date
Initial
2.00
Calibrate pH meter using commercially prepared pH
buffers. Record data below.
pH 4 buffer
manufacturer:
lot number:
expiration date:
2.10
2.20
2.21
2.22
2.30
2.31
2.32
2.40
pH 10 buffer
manufacturer:
lot number:
expiration date:
If using pH strips, test standards and attach below.
pH 4 strip
pH 10 strip
Weigh out 1.74 g potassium phosphate, dibasic
scale tared:
manufacturer:
lot number:
expiration date:
amount used 1.74 g +/- 0.01 g
Measure out 90 ml diH20
Dissolve dibasic potassium phosphate in diH20
potassium phosphate hydrated completely
yes
no
date/time:
Bring total volume to 100ml with diH20
Weigh out 1.36 g potassium phosphate, monobasic
scale tared:
manufacturer:
lot number:
expiration date:
amount used 1.36 g +/- 0.01 g
Measure out 90 ml diH20
Dissolve monobasic potassium phosphate in diH20
potassium phosphate hydrated completely
yes
no
date/time:
Bring total volume to 100ml with diH20
Mix 61 ml dibasic solution with 39 ml monobasic
solution and check pH.
Adjust to pH 7.0 with monobasic solution
pH 7.0 yes
no
Label: 0.1M potassium phosphate buffer, pH 7.0
4
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 2 (page 2 of 2)
Step
Instructions
2.50
Weigh out 0.75 g potassium chloride (KCl)
scale tared:
manufacturer:
lot number:
expiration date:
amount used 0.75 g +/- 0.01 g
Measure out 9 ml potassium phosphate buffer, pH 7.0
Dissolve KCl in 0.1M potassium phosphate buffer, pH 7.0
KCl hydrated completely
yes
no
date/time:
Bring total volume to 10 ml with 0.1M potassium
phosphate buffer, pH 7.0
Label: 1 M KCl, 0.1M potassium phosphate buffer,
pH 7.0
2.51
2.52
Performed by
Date
Initial
5
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 4 – G-50 Size Exclusion Column (page 1 of 2)
Step
Instructions
4.10
Resin Hydration for 8 ml bed volume column
Weigh out 1.0 g Sephadex G-50 resin
scale tared:
manufacturer:
lot number:
expiration date:
amount used 1.0 g +/- 0.01 g
Hydrate using 0.1M potassium phosphate buffer, pH 7.0
Place at 4ºC, overnight
Column Preparation
Pour an 8 ml bed volume G-50 column using a 5 ml
disposable pipette as the column.
Pipette prepared with glass wool stopper in the
end of the pipette and 5 in of tygon tubing for flow
control.
Set up ring stand and clamp. Place column in vertical
position. Clamp off the column and add 3-4 ml 0.1M
potassium phosphate buffer.
Swirl G-50 until the resin is well mixed.
Add slurry to the column using a pasteur pipette and at
the same time open the column.
Do not allow the column to run dry!
Add small aliquots of buffer to keep the fluid surface
above the resin bed.
The pipette will be filled to the lower blue line with
settled resin for a total volume of 8 ml.
Wash the column with 8 ml (one column volume) of
0.1M potassium phosphate buffer. Allow the fluid level
to just reach the top of the resin bed and clamp the
column off.
Protein Separation
Layer 100 μl protein mixture on resin bed
Do not disturb the resin bed!
Unclamp the column and allow the protein mixture to
enter the resin. Clamp off the column.
Wash protein mixture into resin using 100 μl of 0.1M
potassium phosphate buffer (the sample volume) allowing
it to enter the bed. Repeat (total of 2x sample volume of
washes).
Carefully fill reservoir with 0.1M potassium phosphate
buffer, pH 7.0
4.11
4.12
4.20
4.21
4.22
4.23
4.30
4.31
4.32
Performed by
Date
Initial
6
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 4 (page 2 of 2)
Step
Instructions
Performed by
Date
Initial
4.33
Label eight 1.5 ml microfuge tubes (1-8)
Mark 1 ml volume on the outside of each tube with a pen
(Sharpie)
Unclamp the column and collect 1 ml fractions (8 total),
and hold at 4ºC
Do not allow column to run dry while collecting
fractions!
Record color of fractions collected
1.
2.
3.
4.
5.
6.
7.
8.
7
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 5 – Ion Exchange Column (page 1 of 2)
Step
Instructions
5.00
Column Preparation
Obtain preswollen SP-Sepharose
manufacturer:
lot number:
Pour column in Poly-prep disposable column.
Set up ring stand and clamp. Place column in vertical
position. Clamp off the column and add 1 ml 0.1M
potassium phosphate buffer.
Swirl SP-Sepharose until the resin is well mixed.
Add slurry to the column using a pasteur pipette and at
the same time open the column.
Do not allow the column to run dry!
Add small aliquots of buffer to keep the fluid surface
above the resin bed.
The final bed volume should be 1.5 ml of packed resin.
Wash the column with 3 ml (two column volumes) of
0.1M potassium phosphate buffer. Allow the fluid level
to just reach the top of the resin bed and clamp the
column off.
Protein Separation
Layer 100 μl protein mixture on resin bed
Do not disturb the resin bed!
Unclamp the column and allow the protein mixture to
enter the resin. Clamp off the column.
Wash protein mixture into resin using 100 μl of 0.1M
potassium phosphate buffer (the sample volume) allowing
it to enter the bed. Repeat (total of 2x sample volume of
washes).
Carefully layer 2 column volumes (3 ml) of 0.1M
potassium phosphate buffer, pH 7.0 to reservoir on
column
Label three 1.5 ml microfuge tubes
W-1, W-2, and W-3 (mark 1 ml on tubes)
Label three 1.5 ml microfuge tubes
E-1, E-2, and E-3 (mark 1 ml on tubes)
Unclamp column and collect fractions. W-1, W-2 and W3 (1 ml each). Clamp column off.
Carefully layer 2 column volumes of 1M KCl, 0.1M
potassium phosphate buffer, pH 7.0 to reservoir on
column
Unclamp column and collect fractions, E-1, E-2, and E-3
(1 ml each). Clam column off.
Hold both was (W) and elution (E) fractions at 4ºC.
5.01
5.02
5.03
5.10
5.11
5.12
5.13
5.14
5.15
5.16
5.17
Performed by
Date
Initial
8
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 5 (page 2 of 2)
Step
Instructions
Performed by
Date
Initial
5.20
Record any coloration differences in the collected
fractions.
1. W-1
2. W-2
3. W-3
4. E-1
5. E-2
6. E-3
9
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 6 – SDS-PAGE Analysis of Column Fractions (page 1 of 4)
Step
Instructions
6.00
Solution Preparation
Note: If commercially prepared buffers are available, they
may be used in place of the buffers listed below.
Prepare 300 ml of Running Buffer (25 mM Tris, 192 mM
glycine, 0.1% SDS, pH 8.3)
Weight out 0.9 g Trizma-base
manufacturer:
lot number:
expiration date:
molecular weight:
Weight out 4.3 g glycine
manufacturer:
lot number:
expiration date:
molecular weight:
Weight out 1 g SDS
manufacturer:
lot number:
expiration date:
molecular weight:
Measure 10 ml diH2O
Date/time prepared
Chemical fully dissolved YES
NO
LABEL: 10% SDS
Measure 3 ml 10% SDS
Measure 250 ml diH2O and add Trizma-base, glycine and
10% SDS
Date/time prepared
Chemical fully dissolved YES
NO
Bring total volume to 300 ml
LABEL: SDS-PAGE Running Buffer
Prepare 1 ml of Sample buffer (125 mM Tris-HCl, 4%
SDS, 20% glycerol, 10% β-mercaptoethanol)
Prepare a 0.5M Tris-HCl, pH 6.8 solution
manufacturer:
lot number:
expiration date:
molecular weight:
calculation for 0.5M, 20 ml
6.01
date/time prepared
Chemical fully dissolved YES
Performed by
Date
Initial
NO
10
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 6 (page 2 of 4)
Step
Instructions
Performed by
Date
Initial
Prepare 0.04% Bromphenal blue
manufacturer:
lot number:
expiration date:
molecular weight:
calculation for 0.04%, 10 ml
6.02
6.03
date/time prepared
Chemical fully dissolved YES
NO
Solution Preparation
Measure 250 μl 0.5M Tris-HCl, pH 6.8
Measure 400 μl 1-% SDS
Measure 200 μl glycerol
manufacturer:
lot number:
expiration date:
Measure 100 μl β-mercaptoethanol
manufacturer:
lot number:
expiration date:
Measure 50 μl 0.04% Bromphenol blue
Bring total volume to 1 ml
Coomassie blue stain solution
Weigh 2.5 g Coomassie blue R-250
manufacturer:
lot number:
expiration date:
Measure 400 ml methanol
manufacturer:
lot number:
expiration date:
Measure 100 ml glacial acetic acid
manufacturer:
lot number:
expiration date:
Bring total volume to 1 L
Destain Solution
Measure 400 ml methanol
manufacturer:
lot number:
expiration date:
Measure 100 ml glacial acetic acid
manufacturer:
lot number:
expiration date:
Bring total volume to 1 L
11
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 6 (page 3 of 4)
Step
Instructions
6.10
SDS-PAGE: Preparation of gel box and samples
List all materials needed below:
6.20
6.21
Collect column fractions to be assayed on gel
Remove gel from leak-proof pouch
percentage gel:
manufacturer:
lot number:
expiration date:
Remove comb and adhesive strip from gel
Assemble gel in Mini-Protean II cell and fill upper and
lower chambers with Running Buffer (300 ml/box)
Label microfuge tubes and add SDS-sample buffer, 20 μl
per tube
List column fractions assayed:
G-50 fractions: combine 3 and 4, combine 5 and 6
SP-Sepharose fractions: combine W1-3 and E1-3
Add 20 μl amounts of column fraction samples to
appropriate tubes.
1. MW markers (see 6.26)
2. Original protein sample (Load)
3. G-50 fx 3,4
4. G-50 fx 5,6
5. SP-Sepharose W fxs
6. SP-Sepharose E fxs
Prepare tube with molecular weight markers
manufacturer:
lot number:
expiration date:
Poke a hole in the top of each tube and place in heat block
or boiling water bath for 3 min.
Loading samples and running gel
Load samples onto gel using gel loading tops and
micropipettes (refer to Table below)
Connect gel box to power supply and run gel at 150 V
until the bromphenol blue tracking dye reaches the bottom
of the gel (45-50 min)
Visualize proteins in gel
Disassemble gel box and place gel in Coomassie blue
staining solution, microwave 30-45 sec and stain for
6.22
6.23
6.24
6.25
6.26
6.27
6.30
6.31
6.40
Performed by
Date
Initial
12
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
additional 15 min
13
Research
Process Development
Manufacturing
Quality Control
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 6 (page 4 of 4)
Step
Instructions
6.41
Transfer gel to Destain solution, microwave 30-45 sec
and destain 30 min – 1hr
Transfer gel to water to continue destaining until the
background is clear
Dry gel according to instructions included with drying
frame and cellophane sheets and append to SOP.
6.42
6.43
Label
MW
L (load)
G-50 fx 3,4
G-50 fx 5,6
SP-S W
SP-S E
G-50 fx 3,4
G-50 fx 5,6
SP-S W
SP-S E
Amount of sample
5 μl
2 μl
20 μl
20 μl
20 μl
20 μl
20 μl
20 μl
20 μl
20 μl
Performed by
Date
Initial
Amount of SDS-sample buffer
2 μl
20 μl
20 μl
20 μl
20 μl
20 μl
20 μl
20 μl
20 μl
14
Load onto gel
5 μl
4 μl
15 μl
15 μl
15 μl
15 μl
15 μl
15 μl
15 μl
15 μl
Verified by
Date
Initial
Effective: January 17, 2005
Approved:
Mark Lee
Ann Spaulding
Steve Johnson
Sharon Martinez
Research
Process Development
Manufacturing
Quality Control
Section 7 – Comments regarding Standard Operating Procedure: Ultra Hair Protein Additive
Batch Record 1214
7.1
Document below all abnormalities observed as you followed the SOPs for Ultra Hair Protein Additive
15