This document provides instructions and recipes for preparing reagents and performing a western blot analysis. It lists the necessary accessories which include gel components, protein markers, buffers, antibodies, and an ECL kit. Detailed procedures are provided for making separation and stacking gels, preparing protein samples, running the gel electrophoresis, transferring proteins to a membrane, blocking and incubating with primary and secondary antibodies, developing the blot, and capturing results. Precise amounts of reagents are specified for replicating the multi-step process of western blotting.
The technique of paper electrophoresis is simple and inexpensive and requires only micro quantities of plasma for separation.
The support medium is a filter paper
The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution, through which electric current is passed.
Frequently used in isolating proteins, amino acids and oligopeptides.
Preparation of the LAB reagents
How to prepare:
For the Qualitative analysis (identification) of anions and cations from inorganic salt solutions.
1. For sulphate (SO42¯) identification
a. Dilute (6M) Hydrochloric acid (HCl)
b. 0.1M Barium chloride (BaCl2) or Barium nitrate {Ba(NO3)2¬} solution
2. For halide ions identification
a. Dilute nitric acid (2M)
b. 5% Silver nitrate (AgNO3) solution
c. Dilute ammonium hydroxide solution (2M)
d. Concentrated ammonium hydroxide solution (~9M)
3. For nitrate ion identification
a. Concentrated Hydrochloric acid (37%) {÷Conc. Sulphuric acid }
b. 5% Ferrous sulphate (FeSO4) solution
4. For Acetate ion identification
a. Dilute Hydrochloric acid
b. 0.2M Ferric chloride (FeCl3) solution
A. For group I cations
a. Dilute hydrochloric acid (6M)
b. Ammonia solution (9M)
c. Potassium chromate (1M)
B. For group II cations
a. Nitric acid (6M)
b. Stannous chloride (0.1M)
c. Concentrated Ammonia solution (9M)
d. Dilute Ammonia solution (2M)
e. Dilute Sodium hydroxide solution (2M)
C. For group III cations
a. Ammonia solution (9M)
b. Ammonia solution (2M)
c. Potassium ferricyanide (K3[Fe(CN)6]) [250 mg in 10 mL]
d. Potassium thiocyanate [250 mg in 10 mL]
e. Sodium hydroxide (2M)
f. Sodium hydroxide (10M)
The technique of paper electrophoresis is simple and inexpensive and requires only micro quantities of plasma for separation.
The support medium is a filter paper
The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution, through which electric current is passed.
Frequently used in isolating proteins, amino acids and oligopeptides.
Preparation of the LAB reagents
How to prepare:
For the Qualitative analysis (identification) of anions and cations from inorganic salt solutions.
1. For sulphate (SO42¯) identification
a. Dilute (6M) Hydrochloric acid (HCl)
b. 0.1M Barium chloride (BaCl2) or Barium nitrate {Ba(NO3)2¬} solution
2. For halide ions identification
a. Dilute nitric acid (2M)
b. 5% Silver nitrate (AgNO3) solution
c. Dilute ammonium hydroxide solution (2M)
d. Concentrated ammonium hydroxide solution (~9M)
3. For nitrate ion identification
a. Concentrated Hydrochloric acid (37%) {÷Conc. Sulphuric acid }
b. 5% Ferrous sulphate (FeSO4) solution
4. For Acetate ion identification
a. Dilute Hydrochloric acid
b. 0.2M Ferric chloride (FeCl3) solution
A. For group I cations
a. Dilute hydrochloric acid (6M)
b. Ammonia solution (9M)
c. Potassium chromate (1M)
B. For group II cations
a. Nitric acid (6M)
b. Stannous chloride (0.1M)
c. Concentrated Ammonia solution (9M)
d. Dilute Ammonia solution (2M)
e. Dilute Sodium hydroxide solution (2M)
C. For group III cations
a. Ammonia solution (9M)
b. Ammonia solution (2M)
c. Potassium ferricyanide (K3[Fe(CN)6]) [250 mg in 10 mL]
d. Potassium thiocyanate [250 mg in 10 mL]
e. Sodium hydroxide (2M)
f. Sodium hydroxide (10M)
it is a short ppt. on Electrophoresis. Which gives it's defination, Types of electrophoresis ; Like:- Zone electrophoresis, Isoelectric focussing, Immunoelectrophoresis. and their images.
A pipette (also called a point or a pipettor) is a laboratory instrument used to transfer a measured volume of liquid.
Pipettes are commonly used in chemistry and molecular biology research as well as clinical biochemistry tests.
Pipettes come in several designs for various purposes with different levels of accuracy and precision, from single piece flexible plastic transfer pipettes to more complex adjustable or electronic pipettes.
A pipette works by creating a vacuum above the liquid-holding chamber and selectively releasing this vacuum to draw and dispense liquid.
Glassware apparatus and their uses(pdf)Science Equip
This slideshow will give you information about the different types of glassware apparatus and also about its uses. For more visit https://www.scienceequip.com.au/glassware/
The present document provide the information about Methods of extraction of drugs from the biological matrix (protein precipitation method, liquid extraction)
Introduction
History
Elecrophoresis
Principle
Types of electrophoresis
Application
Conclusion
Reference
When a potential difference is applied between the two electrodes in a colloidal solution, It has been observed that the colloidal particles are carried to either the positive or negative electrode.
In other words , they behave as if they are electrically charged w.r.t. the dispersion medium. This phenomenon is known as electrophoresis.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions.
Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
it is a short ppt. on Electrophoresis. Which gives it's defination, Types of electrophoresis ; Like:- Zone electrophoresis, Isoelectric focussing, Immunoelectrophoresis. and their images.
A pipette (also called a point or a pipettor) is a laboratory instrument used to transfer a measured volume of liquid.
Pipettes are commonly used in chemistry and molecular biology research as well as clinical biochemistry tests.
Pipettes come in several designs for various purposes with different levels of accuracy and precision, from single piece flexible plastic transfer pipettes to more complex adjustable or electronic pipettes.
A pipette works by creating a vacuum above the liquid-holding chamber and selectively releasing this vacuum to draw and dispense liquid.
Glassware apparatus and their uses(pdf)Science Equip
This slideshow will give you information about the different types of glassware apparatus and also about its uses. For more visit https://www.scienceequip.com.au/glassware/
The present document provide the information about Methods of extraction of drugs from the biological matrix (protein precipitation method, liquid extraction)
Introduction
History
Elecrophoresis
Principle
Types of electrophoresis
Application
Conclusion
Reference
When a potential difference is applied between the two electrodes in a colloidal solution, It has been observed that the colloidal particles are carried to either the positive or negative electrode.
In other words , they behave as if they are electrically charged w.r.t. the dispersion medium. This phenomenon is known as electrophoresis.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations or anions.
Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
The western blot technique uses gel electrophoresis to separate proteins in a tissue homogenate or extract by molecular weight. The separated proteins on the gel are then transferred to a membrane (usually nitrocellulose or PVDF) which is then incubated with an antibody specific for a target protein. The protein of interest can be visualised using conjugated secondary antibodies and detection reagents.
Do you have a technical question? Get in touch: info@stjohnslabs.com
Protocol series - http://www.stjohnslabs.com/services/video-protocol-series
DNA extraction is an important step in molecular assays and plays a vital role in obtaining highresolution results in gel-based systems, particularly in the case of cereals with high content of interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA extraction method, optimized for rice, which was achieved via creating some modifications in present DNA extraction methods, especially in first step of breaking down and lyses of cell wall, and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal quality and quantity was obtained by the method. The PCR based assays also revealed the efficiency of the method.
The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every laboratory.
RNA, DNA Isolation and cDNA synthesis.pptxASJADRAZA10
Isolation, quantification of nucleic acids from wheat and synthesis of cDNA.
Introduction
List of Genotypes
DNA Isolation (CTAB method)
Qualitative check of DNA- Gel electrophoresis
Quantitative test of DNA- Spectrophotometer
Protocol for RNA Isolation
RNA Confirmation
Normalization of RNA
cDNA Synthesis
Protocol for DNA Isolation of plant
50-100mg (2-3) young leaves were collected, then washed with tap water followed by distilled water in petri dish.
Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by taking 1mL extraction buffer.
1mL (1000μL) of extraction buffer was again added to collect paste from mortar pestle & then transferred to the 2 mL micro centrifuge tube.
The sample in the tube is incubated at 65°C in water bath for 35-45 mins. (Contents in the tube was mixed by inverting at an interval for 5-10 mins)
The tubes were cooled for 10 minutes in ice.
The sample of equal vol (2mL) was centrifuged @14,000 rpm for 10 mins.
After that the supernatant was transferred to new 2 mL centrifuge tube and equal volume (as of sample) of chloroform: Isoamyl alcohol (24:1) was added.
Then mixed gently for 5-7 mins by inverting the tubes.
Again centrifuged for 10 mins @10,000 rpm
After centrifugation, three layers were observed in the tube.
a) aqueous phase i.e. DNA+RNA
b) protein coagulate
c) organic phase i.e. Chloroform
Again the supernatant (aqueous phase) was collected in 1.5mL tube and equal volume of ice-cold isopropanol was added and stored in -20°C overnight.
Following day, tubes were again centrifuged @10,000rpm for 10 mins.
The supernatant was discarded without disturbing the DNA pellet.
70% ethanol is taken and 0.5mL of it was added to the sample and mixed by tapping for 5 mins.
Again centrifuged @10,000rpm for 10 mins and the supernatant was discarded.
Pellet (DNA Precipitate) was air dried for 10 mins.
Then dissolved in 50μL TE-1X Buffer and the sample was stored at -20°C.
1g of analytical grade Agarose was weighed.
100 mL of autoclaved 1X TBE was added in flask.
Now heated on the oven until the solution becomes transparent.
Solution was allowed to cool down to 60℃.
2 μL of Ethidium Bromide (EtBr) is added in the flask.
Melted agarose gel was poured into the casting tray along with comb.
Any bubble in the gel was removed.
After solidification of gel, comb was removed gently and then running buffer was added in the electrophoretic tank.
Once gel got solidified, it was transferred it into gel tank.
A parafilm was taken and on it 2μL loading dye and 3μL sample was taken, gently mixed with the pipette tip only.
Then the mixture (sample +loading dye) was loaded into the well.
Then electrophoretic unit was run at 90 volt for 50-55 mins.
After that gel was put into the Gel Doc to see the DNA band
(using UV light).
Bright colour band were observed as in the figure.
Few (100-150mg) young leaves were ground into fine powder using liquid Nitrogen.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
1. Accessories for western blotting
1. Gel Accessories
a) Ammonium per sulfate
b) TRIS PH 6.8 (1M)
c) TRIS PH 8.8 (1.5)
d) Temed
e) Acrlamide/Bis (30:80)
2. Protein marker
3. 4X buffer
4. Running buffer
5. Transfer buffer
6. 20X Ponceau Stain
7. 5% Non-Fat Milk.
8. Primary Antibody
9. Secondary Antibody
10. 10X TBS
11. 1X TBST
12. ECL-Kit (Enhanced Chemiluminescence-Kit)
1.a Ammonium per sulfate
Take 0.14g/140mg Ammonium per sulfate and dissolve it in distilled water, making final volume 1ml.
1.b TRIS PH 6.8 (1M)
First prepare 0.2M EDTA and, 10% SDS.
0.2M EDTA (200 ml) = Take 11.6896g of EDTA and dissolve in distilled water by magnetic stirring, making
final volume 200ml.
SDS 10% (250 ml) = Take 25g SDS; dissolve in 100ml of distilled water by magnetic stirring. Color
becomes transparent upon final dissolution. Then make final volume 250ml with the distilled water.
Now go further to prepare 1M, 200ml TRIS. So take 24.228gm of TRIS. Dissolve in 100ml of water. Adjust
PH 6.8. Add 8ml of 0.2M EDTA and 10% SDS each. Make final volume 200ml with distilled water. So it
will be 1M, 200ml TRIS, PH 6.8, having 8ml 0.2M EDTA and 10% SDS each.
1.c TRIS PH 8.8 (1.5M)
Prepare 0.2M EDTA and, 10% SDS as above.
2. Now go further to prepare 1.5M, 250ml TRIS. Take 45.4275gm of TRIS. Dissolve in 100ml of water.
Adjust PH 8.8. Add 10ml of 0.2M EDTA and 10% SDS each. Make final volume 250ml with distilled water.
So it will be 1.5M, 250ml TRIS, PH 8.8, having 10ml 0.2M EDTA and 10% SDS each.
1.d Temed
We buy it ready made from company.
1.e Acrylamide/Bis (30 : 0.8)
We make 1 liter solution. For this we take 300g= Acrylamide, and 8g= Bis. Dissolve in 500ml of distilled
water. Then make final volume 1000ml. Filter the solution in clean bench and store at 4°C.
2. Protein Marker
Std. Protein Marker:1X buffer
1:50 (e.g 50ul & 950ul)
3. 4X buffer
1M Tris (PH 6.8)---------- 1.25ml
Glycerol-------------------- 4ml
β- mercaptoethanol---- 2ml
1% BPB-------------------- 0.25ml
D. Water------------------ 2.5ml
SDS------------------------- 0.8g
--------------------------------------------------------------
Total----------------------- 10ml
4. Running buffer
We prepare 5X Tris running buffer, 2 liter.
Tris= 30g, Dissolve in 500 ml of distilled water. Then make the volume 2 liters
SDS= 10g, with the distilled water.
Glycine= 144g
5. Transfer buffer
We make 1 liter of transfer buffer. It contains:
100mM CAPS PH 11= 100ml,
Methanol= 100ml,
Distilled water= 800ml.
Whereas,
3. 100mM CAPS= 22.132g CAPS/liter of distilled water, as;
1000mM/1M CAPS= 221.32g/ liter of distilled water.
1M= 1000mM
100mM= 1000mM/10, SO, 221.32/10=22.132g
6. 20X Ponceau Staining Solution
2% (W/V) Ponceau S in 30% Tricholoroacetic acid and 30% sulfosalicylic acid in distilled water. We can
buy it ready made. When using in western blot, we make it 1X by taking 1ml 20X ponceau stain soln. and
adding 19ml distilled water to it or half of these i.e 500ul and 9.5ml.
7. 5% Non-Fat Milk
Non-Fat Milk= 2g powder. Dissolve in 1X TBS, making final volume 40ml with 1X TBS.
8. Primary Antibody
We make it by 1:1000, primary antibody and 1X TBS respectively.
So;
1X TBS---------------------- 10ml
1° Anb---------------------- 10ul
2% Sodium azide-------- 20ul
9. Secondary Antibody
We make it by 1:2000, secondary antibody and 1X TBS respectively.
So;
1X TBS---------------------- 20ml
1° Anb---------------------- 10ul
No sodium azide in this case.
10. 10X TBS
Tris= 48.4g, Nacl= 160g, Distilled water q.s to make 2 liters. When using, we make it 1X by taking 100ml
10X TBS and 900ml distilled water.
4. 11. 1X TBST
1 liter 1X TBS, and add 1ml tweens-20, or half of these quantities.
12. ECL- Kit (Enhanced Chemiluminescence-Kit)
It contains two solutions. Mix both of these 1:1 and vortex. Ready to apply.
5. Western Blotting
1) Spray 70% ethanol on the glasses to be used in making assembly. Then clean with tissue paper.
2) Set the assembly and fix it in the stand. Add water into it and wait for 5-10 min for checking
either water is leaking or not.
3) When water is not leaking, start making gel according to the specifications given in table below.
We usually make 10% separation gel and 5% stacking gel.
Table. 1 Gel Composition
Separation Gel
Gel %age Acrylamide/Bis
30:0.8
Tris-Base PH 8.8 D.Water APS Temed
7% (1)
7% (2)
7% (4)
1.75ml 1.88ml 3.88ml 3.75ul 75ul
3.5ml 3.75ml 7.75ml 7.5ul 150ul
7ml 7.5ml 15.5ml 15ul 300ul
7.5% (2)
7.5% (4)
3.75ml 3.75ml 7.5ml 7.5ul 150ul
7.5ml 7.5ml 15ml 15ul 300ul
8% (1)
8% (2)
8% (3)
8% (4)
2ml 1.88ml 3.63ml 4ul 75ul
4ml 3.75ml 7.25ml 7.5ul 150ul
6ml 5.63ml 10.88ml 12ul 225ul
8ml 7.5ml 14.5ml 15ul 300ul
10% (1)
10% (2)
10% (3)
10% (4)
2.38ml 1.88ml 3.13ml 4ul 75ul
4.75ml 3.75ml 6.25ml 7.5ul 150ul
7.12ml 5.63ml 9.38ml 12ul 225ul
10ml 7.5ml 12.5ml 15ul 300ul
12% (1)
12% (2)
12% (4)
3ml 1.88ml 2.63ml 3.75ul 75ul
6ml 3.75ml 5.25ml 7.5ul 150ul
12ml 7.5ml 10.5ml 15ul 300ul
12.5% (2)
12.5% (4)
6.25ml 3.75ml 5ml 7.5ul 150ul
12.5ml 7.5ml 10ml 15ul 300ul
15% (2) 7.5ml 3.75ml 3.75ml 7.5ul 150ul
Stacking Gel
Gel %age Acrylamide/Bis
30:0.8
Tris-Base PH 6.8 D.Water APS Temed
3% (2) 1.5ml 3.8ml 8.8ml 15ul 300ul
4.6% (2)
4.6% (4)
4.6% (6)
0.58ml 0.95ml 2ml 4ul 75ul
1.15ml 1.9ml 4ml 7.5ul 150ul
2.3ml 3.8ml 8ml 15ul 300ul
5% (2)
5% (4)
1.2ml 2ml 4.3ml 7.5ul 150ul
2.4ml 4ml 8.6ml 15ul 300ul
4) First make separation gel. For this, in a corning tube, take:
(i) Acrylamide
(ii) Tris-base PH 8.8
(iii) Distilled water
(iv) Ammonium per sulfate, and
(v) Temed.
(vi) Now vortex for few seconds.
Before adding TEMED, make
assembly ready for pouring gel
into it with the help of 5ml Tip.
6. (vii) Pour the gel solution into the assembly up to the Mark present in the stand. Add Iso-
propanol/water in the remaining space for making the upper edge of the gel smooth. Wait
for 20-30 min. In this time gel will be formed.
5) Now remove the water/iso propanol from the assembly, dry the upper empty part of gel with
absorbent paper and insert the appropriate comb just above the upper edge of the gel. Start
making 5% stacking gel in a corning tube. Add:
(i) Acrylamide
(ii) Tris-base PH 6.8
(iii) Distilled water
(iv) Ammonium per sulfate
(v) TEMED.
(vi) Vortex for few seconds and transfer very quickly to the assembly in between the wells
formed over the separating gel by comb with the help of 1ml tip. Take intensive care that no
water bubble is entrapped. Wait 10-15 min for gel formation.
6) During the formation of stacking gel, prepare the sample for loading. We mix protein sample
with 4X buffer and distilled water. And quantity of sample is taken according to the Std. Curve
formula, e.g. y= 0.0648x + 0.0026(Used in MS Excel).
Sample Absorbance 2ug 1ug 20ug Load H2O 4X buffer Total
Control 0.247 3.83 1.91 10.47 4.53 5 20
3 hours 0.251 3.89 1.95 10.25 4.75 5 20
Absorbance= Y values (0.247, 0.251)
2ug(X value from y= 0.0648x + 0.0026) = Qty of sample used in analysis by UV spectrophotometer.
1ug= 2ug/2.
20ug Load= Desirable & variable load. 20/1.91= Loading Qty. divided by first value in 1ug column.
H2O= Subtract the highest value in 20ug column from 15. (Total volume you make).
4X buffer= 1/3 of total volume (15/3=5). Total= Add last three columns.
7) After combining protein sample, distilled water and 4X buffer, give a flash on centrifugator and
then keep the sample for 5 minutes in boiled water.
8) Again give a flash in the centrifugator. Now remove the comb, wash the wells with distilled
water twice, dry with absorbent paper and load the sample in the following manner:
9) Now in running apparatus, add 1X running buffer, and start running at 90 volts for 2-2.5 hours.
Take care of right positions of the electrodes while supplying power, red on red and black on
black. Check visually that running buffer has started upward movement after getting power
supply.
10) After completion of running time, transfer the gel to Nitrocellulose membrane. For this take
Transfer cassette and keep:
1X running
buffer
10-20ul
Protein
Marker
10-20ul
Sample Sample Sample Sample Sample 1X running
buffer
10-20ul
1X running
buffer
10-20ul
7. Sponge Filter paper Gel NC membrane Filter paper Sponge
NC paper size= 9 x 6 inch
11) Close the transfer cassette, add transfer buffer in transfer apparatus, insert the transfer cassette
into it and start the transferring process. Apply 90 volts for 25 minutes, and then change the
positions of cassettes and run for 25 minutes again. Take care of red and black sides.
12) On completion of transferring time, remove the NC paper from cassette, and add to 1X Ponceau
stain solution. It will make protein bands visible. After that wash the NC paper with distilled
water and cut it according to the molecular weight of proteins you need. Protein marker will be
showing you the range of molecular weight. After making the NC strips, wash for 5 minutes in
distilled water and then keep in 5% Non-fat milk at -20°C for 2 hours. This step is called blocking.
13) After blocking step, put the strips in appropriate primary antibody for 12-16 hrs at -20°C.
14) Wash the strips with TBST for 5, 5, 10, 10, and 10 minutes.
15) Apply appropriate secondary antibody for 2 hours keeping at -20°C.
16) Wash the strips with TBST for 5, 5, 10, 10, and 10 minutes.
17) Dry the strips by keeping it on tissue paper and then fix these in X-ray cassette, pour solution
from ECL-Kit (1:1), wait for 2 minutes so that the strips properly absorb the solution, then fix the
X-ray in this cassette in the dark room. (We usually fix 2 X-ray at first, then after taking result of
first one, we fix 1 another X-ray and takes its result within 24 hours).
18) After fixing the X-ray wait for 30-40 minutes. Then develop the X-ray and take the result.
Properly label you results.