A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose, polyvinylidene fluoride (PVDF) or nylon membrane). In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining (for example, silver staining of proteins), autoradiographic visualization of radioactive labelled molecules (performed before the blot), or specific labelling of some proteins or nucleic acids. The latter is done with antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity (and so, the molecules we search in the blot) is visualized by incubation with proper reactive, rendering either a colored deposit on the blot or a chemiluminiscent reaction which is registered by photographic film. Southern blot A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.[1] Western blot A Western blot is used for the detection of specific proteins in complex samples. Proteins are first separated by size using electrophoresis before being transferred to an appropriate blotting matrix (usually PVDF or nitrocellulose) and subsequent detection with antibodies. Eastern blot The Eastern blot is used for the detection of specific posttranslational modifications of proteins. Proteins are separated by gel electrophoresis before being transferred to a blotting matrix whereupon posttranslational modifications are detected by specific substrates (cholera toxin, concanavalin, phosphomolybdate, etc.) or antibodies.

1. 1
Blotting
• Blotting :- is the technique in which nucleic
acids or proteins are immobilized onto a solid
support generally nylon or nitrocellulose
membranes .
‫النوويه‬ ‫االحماض‬ ‫فصل‬ ‫في‬ ‫درسناها‬ ‫اللي‬ ‫االجهزه‬ ‫بستخدم‬ ‫ما‬ ‫بعد‬
‫ال‬ ‫اعمل‬ ‫هبدأ‬ ‫عليهم‬ ‫بيحتوي‬ ‫اللي‬ ‫الجل‬ ‫عندي‬ ‫وهينتج‬ ‫والبروتين‬
Blotting
‫الي‬ ‫الجل‬ ‫في‬ ‫اللي‬ ‫اوالبروتين‬ ‫النوويه‬ ‫االحماض‬ ‫هنقل‬ ‫اني‬ ‫بمعني‬
‫طريقة‬ ‫اكذا‬ ‫طريق‬ ‫عن‬ ‫غشاء‬‫هندرسهم‬‫عشان‬ ‫دي‬ ‫الطريقه‬ ‫وبعمل‬
‫ج‬ ‫احدد‬ ‫عايز‬ ‫بكون‬‫االحماض‬ ‫في‬ ‫عايزه‬ ‫انا‬ ‫معين‬ ‫تتابع‬ ‫او‬ ‫معين‬ ‫ين‬
‫او‬ ‫البروتين‬ ‫بفصل‬ ‫ما‬ ‫وبعد‬ ‫البروتين‬ ‫في‬ ‫معين‬ ‫جزء‬ ‫او‬ ‫النوويه‬
‫حاجه‬ ‫بستخدم‬ . ‫الغشاء‬ ‫علي‬ ‫بنقلها‬ ‫وبعدين‬ ‫الجل‬ ‫في‬ ‫النوويه‬ ‫االحماض‬
‫يظهرلي‬ ‫عايزه‬ ‫انا‬ ‫اللي‬ ‫المعين‬ ‫التتابع‬ ‫مع‬ ‫تتفاعل‬ ‫عشان‬ ‫بروب‬ ‫اسمها‬
‫والبروب‬ ‫لألشعة‬ ‫الغشاء‬ ‫اعرض‬ ‫لما‬‫المعين‬ ‫التتابع‬ ‫مع‬ ‫تفاعل‬ ‫بيعمل‬ ‫دا‬
‫هيدروجينيه‬ ‫روابط‬ ‫ويكون‬ ‫بيه‬ ‫بيرتبط‬ ‫يعني‬ ‫تهجين‬ ‫وبيعمل‬ ‫دا‬‫مع‬
‫النيتروجينيه‬ ‫القواعد‬‫عايزه‬ ‫انا‬ ‫اللي‬ ‫بالتتابع‬ ‫يرتبط‬ ‫ما‬ ‫وبمجرد‬ ‫معاه‬
‫المحاضرة‬ ‫فكره‬ ‫تعتبر‬ ‫ودي‬ ‫لألشعة‬ ‫بعرضه‬ ‫لما‬ ‫الشاشه‬ ‫في‬ ‫بيظهرلي‬
. ‫النهاردة‬

2. 2
There are different blotting procedures
depending on the type of molecule being
transferred:-
• When DNA fragments are
transferred the procedure is called a Southern
blot, named after the person who first developed
it, Edward Southern.
•With Northern blotting, RNA molecules are
transferred
•and with Western blotting, protein molecules are
transferred.

3. 3
Probe
◘ A probe (a piece of nucleic acid with identical
and specific sequence to the organism or gene of
interest) can then hybridize (join) to the
biological molecules (DNA, RNA or protein) with
an identical sequence on the membrane.
◘ The hybridization between the blotted DNA
and probe is visualized by labelling the probe in
some way.

4. 4
DNA probe joins together (hybridizes) with target DNA
blotted on a membrane.
General principle
•All blotting procedures begin with a standard process
called gel electrophoresis. During this step, DNA,
RNA, or proteins are loaded on to an agarose or
polyacylamide gel (that functions like a molecular
sieve) and are then run through an electric field.
•Transfer is initiated when the nylon or nitrocellulose
membrane is laid on top of the gel and biological
molecules are transferred from the gel to the
membrane .
‫ا‬ ‫تتنقل‬ ‫بتبدأ‬‫فوق‬ ‫محطوط‬ ‫بيبقي‬ ‫اللي‬ ‫الغشاء‬ ‫الي‬ ‫الجل‬ ‫من‬ ‫النوويه‬ ‫الحماض‬
‫الجل‬
•It is used to verify the presence or absence of a
specific nucleotide sequence in DNA from different
sources .
‫انا‬ ‫معين‬ ‫تتابع‬ ‫غياب‬ ‫او‬ ‫وجود‬ ‫احدد‬ ‫عشان‬ ‫اصال‬ ‫دا‬ ‫الموضوع‬ ‫بستخدم‬
‫عايزه‬
• DNA is isolated from each source and then digested
with a specific restriction enzyme .

5. 5
• The DNA restriction fragments are loaded onto an agarose
gel and the fragments separated by electrophoresis
according to size .
• The fragments visible with Ethidium bromide under UV-
light
• The DNA is then transferred from the agarose gel to a
membrane ( nylon or nitrocellulose ) and denatured to
produce single strands .
‫تكامل‬ ‫يعمل‬ ‫وعشان‬ ‫الغشاء‬ ‫علي‬ ‫مزدوج‬ ‫شريط‬ ‫بيبقي‬ ‫ايه‬ ‫ان‬ ‫الدي‬ ‫بستخدم‬ ‫لما‬ ‫طبعا‬
‫وتهجين‬‫لشريط‬ ‫يتحول‬ ‫الزم‬ ‫البروب‬ ‫وبين‬ ‫بينه‬ ‫ما‬‫االول‬ ‫الزم‬ ‫كدا‬ ‫عشان‬ ‫مفرد‬
‫مفرد‬ ‫لشريط‬ ‫يتحول‬
• A nucleic acid probe is labeled , usually by incorporating
radioactivity or tagging the molecule with a fluorescent dye .
‫ال‬ ‫الزم‬‫برو‬‫عشا‬ ‫مشعة‬ ‫صبغة‬ ‫او‬ ‫مشعة‬ ‫بماده‬ ‫متعلم‬ ‫يكون‬ ‫ب‬‫احطه‬ ‫لما‬ ‫مكانه‬ ‫احدد‬ ‫ن‬
‫الشاشه‬ ‫تحت‬
• The labeled probe is added and it binds to complementary
DNA on the membrane .
• To detect the position of the labeled probe , the membrane
is covered with an x-ray film and after development the
position of the probe becomes visible.
‫للجهاز‬ ‫وانقله‬ ‫راي‬ ‫لالكس‬ ‫فيلم‬ ‫علي‬ ‫الغشاء‬ ‫بنقل‬ ‫دا‬ ‫فات‬ ‫اللي‬ ‫كل‬ ‫بخلص‬ ‫ما‬ ‫اول‬
‫ارتباط‬ ‫نتيجه‬ ‫طالعة‬ ‫اللي‬ ‫المشعة‬ ‫الماده‬ ‫هيظهرلي‬ ‫اللي‬) ‫تهجين‬ (‫انا‬ ‫اللي‬ ‫التتابع‬
. ‫البروب‬ ‫مع‬ ‫عايزه‬

6. 6

7. 7
• This Technique is used to study gene expression by
detection of RNA ( or isolated mRNA ) in a sample .
• with northern blotting it is possible to observe cellular
control over structure and function by determining the
particular gene expression levels during differentiation ,
morphogenesis , as well as abnormal or diseased
conditions .
Northern blotting
• starting with extraction of total RNA from a
homogenized tissue sample .
• RNA separated by gel electrophoresis .
• A nylon membrane with a positive charge is the
most effective for use in northern blotting since
the negatively charged nucleic acids have a high
affinity for them .
• The transfer buffer contains Formamide
because it lowers the annealing temperature of
the probe – RNA interaction preventing RNA
degradation by high temperatures .

8. 8
• After the probe has been labeled , it is hybridized
to the RNA on the membrane .
• The membrane is washed to ensure that the
probe has bound specifically .
• The hybrid signals are then detected by X-ray
film and can be quantified by densitometry .
Advantages & Disadvantages of Northern
blotting
• Northern blotting is able to detect small changes
in gene expression that microarrays cannot
• A problem in Northern blotting is often sample
degradation by RNases ( Both endogenous to the
sample and through through environmental
contamination ) .

9. 9
Western blotting
• ◘ • Steps :-
1- Sample preparation .
2- Electrophoresis .
3- Transfer.
4- Blocking .
5- Detection.
‫بقي‬ ‫هنبدأ‬ ‫دي‬ ‫قبل‬ ‫اللي‬ ‫المحاضرات‬ ‫في‬ ‫خطوتين‬ ‫اول‬ ‫اخدنا‬ ‫احنا‬
. ‫الثالثة‬ ‫الخطوة‬ ‫من‬ ‫الوقتي‬
◘ Transfer
• To make The proteins accessible to antibody
detection , they are moved from within the gel
onto a nitrocellulose or polyvinylidene
difluoride ( PVDF ) membrane similar to
Southern blot DNA transfer .
‫البر‬ ‫ان‬ ‫هيكون‬ ‫هنا‬ ‫االختالف‬ ‫ولكن‬ ‫فاتت‬ ‫اللي‬ ‫الطريقة‬ ‫نفس‬‫وب‬
‫هيبقي‬
( Anti body )

10. 10
• Another method for transferring the
proteins is called electro blotting and uses
an electric current to pull proteins from the
gel into the PVDF or nitrocellulose
membrane .
• As a result of this “ blotting “ process , the
proteins are exposed on a thin surface layer
for detection .
• Protein binding is based upon
hydrophobic interaction , as well as
charged interactions between the
membrane and protein.
• Nitrocellulose membrane are cheaper
than PVDF , but are far more Fragile and
do not stand up well to repeated probings.
‫هشاشه‬ ‫اكثر‬ ‫بيكون‬

11. 11
Blocking
• Steps must be taken to prevent interactions between the
membrane and the antibody used for detection of the target
protein .
• Blocking of non-specific binding is achieved by placing the
membrane in a dilute solution of protein , typically Bovine
serum albumin ( BSA) or non-fat dry milk ( both are
inexpensive ) , with a minute ( percentage of detergent such
as Tween 20 .

12. 12
Detection
Two steps :-
1- ◘ primary Antibody
• After blocking a diluted solution of
primary antibody ( generally between
0.5 and 5 micrograms/ml ) is
incubated with the membrane under
gentle agitation .
• The solution is composed of
buffered saline solution with a small
percentage of detergent , and
sometimes with powdered milk or
BSA .
• The Antibody solution and the
membrane can be incubated together
from 30 minutes to Overnight .

13. 13
2- ◘ Secondary Antibody
• After rinsing the membrane to remove unbound
primary antibody .
‫وبين‬ ‫بينها‬ ‫ما‬ ‫تفاعل‬ ‫هيحصل‬ ‫االولية‬ ‫المضادة‬ ‫االجسام‬ ‫بضيف‬ ‫لما‬
‫مع‬ ‫دي‬ ‫االجسام‬ ‫تفاعل‬ ‫بيمنع‬ ‫بلوكنج‬ ‫بضيف‬ ‫كدا‬ ‫عشان‬ ‫الغشاء‬
‫االجسام‬ ‫بسيب‬ ‫كدا‬ ‫وبعد‬ ‫بس‬ ‫البروتين‬ ‫مع‬ ‫تتفاعل‬ ‫ويخليها‬ ‫الغشاء‬
‫الغشاء‬ ‫هغسل‬ ‫وبعدين‬ ‫معين‬ ‫لوقت‬ ‫البروتين‬ ‫مع‬ ‫تتفاعل‬ ‫المضاده‬
‫ا‬ ‫المضادة‬ ‫االجسام‬ ‫اضيف‬ ‫واؤجع‬‫االجسام‬ ‫في‬ ‫هتشبك‬ ‫اللي‬ ‫لثانوية‬
‫األولية‬ ‫المضادة‬
• The membrane is exposed to another antibody ,
directed at a specific portion of the primary
antibody .
• This is known as secondary antibody , and due
to its targeting properties , tends to be referred to
as “ Antimouse” “ Anti-goat “ etc ,
- Antibodies come from animal sources ( or
animal sourced hybridoma cultures ) ; an
anti-mouse secondary will bind to just about
any mouse-sourced primary anti body

14. 14
• The secondary antibody is usually linked to
biotin or to a reporter enzyme such as alkaline
phosphatase or horseradish peroxidase .
• This means that several secondary antibodies
will bind to one primary antibody and enhance
the signal .

15. 15
Eastern blotting
• it is a technique to detect protein post
translational modification and is an extension of
the biochemical technique of western blotting .
‫المحاضرة‬ ‫تمت‬
Q.A
‫لما‬ ‫اال‬ ‫هتتفهم‬ ‫مش‬ ‫عشان‬ ‫تذاكروا‬ ‫ما‬ ‫بعد‬ ‫ليها‬ ‫فيديوهات‬ ‫تسمعوا‬ ‫ياريت‬
‫االول‬ ‫بعينيكم‬ ‫تشوفوها‬☺

16. 16